Compositions including triciribine and methods of use thereof

ABSTRACT

This invention encompasses combination therapies including TCN, TCN-P, TCN-PM and/or related compounds and one or more additional anti-cancer agents, for example, taxanes a molecule that modulates the HER2/neu (erbB2) receptor, anthracyclin compounds, epidermal growth factor receptor inhibitor compounds, one or more platinum compounds and bortezomib and derivatives thereof and compositions with reduced toxicity for the treatment and prevention of tumors, cancer, and other disorders associated with abnormal cell proliferation.

The present application is a continuation of U.S. patent applicationSer. No. 12/992,556, filed May 18, 2011, now abandoned which is acontinuation-in-part of U.S. patent application Ser. No. 12/118,848,filed May 12, 2008, which is abandoned, and is a continuation-in-part ofU.S. patent application Ser. No. 12/118,861, filed May 12, 2008, and isabandoned, and is a continuation-in-part of U.S. patent application Ser.No. 12/118,868, filed May 12, 2008, and is abandoned, and is acontinuation-in-part of U.S. patent application Ser. No. 12/118,870,filed May 12, 2008, and is abandoned, and is a continuation-in-part ofU.S. patent application Ser. No. 12/118,834, filed May 12, 2008, and isabandoned, and is a continuation-in-part of U.S. patent application Ser.No. 12/118,828, filed May 12, 2008, and is abandoned, and is acontinuation-in-part of U.S. patent application Ser. No. 11/096,082,filed Mar. 29, 2005, now U.S. Pat. No. 8,435,959 which claims thebenefit of U.S. provisional patent application No. 60/557,599, filedMar. 29, 2004, the disclosures of each of which is incorporated hereinby reference in their entireties.

1. ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This invention was made, in part with government support under grant(contract) nos. P30-CA 16672; 1RO1-CA 109570; 1RO1-CA 119127;PO1-CA099031 project 4; and P50 CA116100 01 project 4 awarded by theNational Institutes of Health and grant no. DAMD17-02-1-0694 awarded bythe Department of Defense. The government has certain rights to thisinvention.

2. FIELD OF THE INVENTION

The invention encompasses combination therapies including TCN, TCN-P,TCN-PM and/or related compounds and one or more additional anti-canceragents for example, one or more taxanes; a molecule that modulates theHER2/neu (erbB2) receptor, for example, trastuzumab or a salt thereof;anthracyclin compounds; epidermal growth factor receptor inhibitorcompounds, particularly erlotinib-like compounds; one or more platinumcompounds; and bortezomib or salts thereof. The invention alsoencompasses compositions with reduced toxicity for the treatment andprevention of tumors, cancer, and other disorders associated withabnormal cell proliferation.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format, created Nov. 3, 2005, is named 065031-5001-USSequence Listing.txt, and is hereby incorporated by reference in itsentirety.

3. BACKGROUND OF THE INVENTION

Cancer is an abnormal growth of cells. Cancer cells rapidly reproducedespite restriction of space, nutrients shared by other cells, orsignals sent from the body to stop reproduction. Cancer cells are oftenshaped differently from healthy cells, do not function properly, and canspread into many areas of the body. Abnormal growths of tissue, calledtumors, are clusters of cells that are capable of growing and dividinguncontrollably. Tumors can be benign (noncancerous) or malignant(cancerous). Benign tumors tend to grow slowly and do not spread.Malignant tumors can grow rapidly, invade and destroy nearby normaltissues, and spread throughout the body.

Cancers are classified according to the kind of fluid or tissue fromwhich they originate, or according to the location in the body wherethey first developed. In addition, some cancers are of mixed types.Cancers can be grouped into five broad categories, carcinomas, sarcomas,lymphomas, leukemias, and myelomas, which indicate the tissue and bloodclassifications of the cancer. Carcinomas are cancers found in bodytissue known as epithelial tissue that covers or lines surfaces oforgans, glands, or body structures. For example, a cancer of the liningof the stomach is called a carcinoma. Many carcinomas affect organs orglands that are involved with secretion, such as breasts that producemilk. Carcinomas account for approximately eighty to ninety percent ofall cancer cases. Sarcomas are malignant tumors growing from connectivetissues, such as cartilage, fat, muscle, tendons, and bones. The mostcommon sarcoma, a tumor on the bone, usually occurs in young adults.Examples of sarcoma include osteosarcoma (bone) and chondrosarcoma(cartilage). Lymphoma refers to a cancer that originates in the nodes orglands of the lymphatic system, whose job it is to produce white bloodcells and clean body fluids, or in organs such as the brain and breast.Lymphomas are classified into two categories: Hodgkin's lymphoma andnon-Hodgkin's lymphoma. Leukemia, also known as blood cancer, is acancer of the bone marrow that keeps the marrow from producing normalred and white blood cells and platelets. White blood cells are needed toresist infection. Red blood cells are needed to prevent anemia.Platelets keep the body from easily bruising and bleeding. Examples ofleukemia include acute myelogenous leukemia, chronic myelogenousleukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia.The terms myelogenous and lymphocytic indicate the type of cells thatare involved. Finally, myelomas grow in the plasma cells of bone marrow.In some cases, the myeloma cells collect in one bone and form a singletumor, called a plasmacytoma. However, in other cases, the myeloma cellscollect in many bones, forming many bone tumors. This is called multiplemyeloma.

Thus, a combination of a TCN, TCN-P, TCN-PM and/or related compounds ora derivative thereof and an additional anti-cancer agent holds promiseas a potential combination therapy for treating tumors, cancer, andabnormal cell proliferation.

3. SUMMARY OF THE INVENTION

The invention encompasses novel therapeutic regimens of triciribine(“TCN”), triciribine phosphate (“TCN-P”), triciribine phosphatemonohydrate (“TCN-PM”) and related compounds in combination with one ormore additional anti-cancer agents, for example, one or more taxanes, amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof; anthracyclin compounds, epidermal growthfactor receptor inhibitor compounds, particularly erlotinib-likecompounds, one or more platinum compounds and bortezomib or saltsthereof.

The invention is based on the discovery that tumors or cancers, whichoverexpress Akt kinase are sensitive to the cytotoxic effects of TCN,TCN-P, TCN-PM and related compounds and a synergistic affect arises whenadministering a combination of these compounds and certain additionalanti-cancer agents. The inventors have determined, contrary to the priorknowledge and experience, how to successfully use a combination of TCN,TCN-P, TCN-PM and related compounds and at least one additionalanti-cancer agent to treat tumors and cancer by one or a combination of(i) administering TCN, TCN-P, TCN-PM and related compounds and at leastone additional anti-cancer agent to patients who exhibit enhancedsensitivity to the triciribine; (ii) use of a described dosage levelthat minimizes the toxicity of the drugs but vet still exhibitsefficacy; or (iii) use of a described dosage regimen that minimizes thetoxicity of the drugs.

The inventions also encompasses methods useful to treat tumors andcancers that are particularly susceptible to the toxic effects of TCN,TCN-P, TCN-PM and/or related compounds. In another embodiment, methodsare provided for treating a tumor in a mammal, particularly a human thatincludes (i) obtaining a biological sample from the tumor; (ii)determining whether the tumor overexpresses an Akt kinase, and (iii)treating the tumor that overexpresses Akt kinase with TCN, TCN-P, TCN-PMand/or related compounds in combination with one or more additionalanti-cancer agents, as described herein.

In another embodiment, the level of Akt kinase expression can bedetermined by assaying the tumor or cancer for the presence of aphosphorylated Akt kinase, for example, by using an antibody that candetect the phosphorylated form. In another embodiment, the level of Aktexpression can be determined by assaying a tumor or cancer cellsobtained from a subject and comparing the levels to a control tissue. Incertain embodiments, the Akt can be overexpressed at least 2, 2.5, 3 or5 fold in the cancer sample compared to the control. In certainembodiments, the overexpressed Akt kinase can be a hyperactivated andphosphorylated Akt kinase.

In another aspect of the invention, dosing regimens are provided thatlimit the toxic side effects of TCN, TCN-P, TCN-PM and/or relatedcompounds in combination with one or more additional anti-cancer agents,as described herein. In another embodiment, such dosing regimensminimize or eliminate toxic side effects, including, but not limited to,hepatoxicity, thrombocytopenia, hyperglycemia, vomiting, hypocalcemia,anemia, hypoalbunemia, myelosuppression, hypertriglyceridemia,hyperamylasemia, diarrhea, stomachitis and/or fever.

In another embodiment, the administration of TCN, TCN-P, TCN-PM and/orrelated compounds in combination with one or more additional anti-canceragents, as described herein, provides at least a partial, such as atleast 15, 20 or 30%, or complete response in vivo in at least 15, 20, or25% of the subjects.

In another embodiment, a method is provided to treat a subject which hasbeen diagnosed with a tumor by administering to the subject an effectiveamount of TCN, TCN-P, TCN-PM and/or related compounds in combinationwith one or more additional anti-cancer agents, as described hereinaccording to a dosing schedule that includes administering the drugapproximately one time per week for approximately three weeks followedby a one week period wherein the drug is not administered.

In another embodiment, methods are provided to treat tumor or cancer ina subject by administering to the subject a dosing regimen of 10 mg/m²or less of TCN, TCN-P, TCN-PM and/or related compounds in combinationwith one or more additional anti-cancer agents, as described herein eachone time per week. In another embodiment, the TCN, TCN-P, TCN-PM and/orrelated compounds in combination with one or more additional anti-canceragents, as described herein can be administered as a single bolus doseover a short period of time, for example, about 5, 10 or 15 minutes.

In further embodiments, dosing schedules are provided in which the TCN,TCN-P, TCN-PM and/or related compounds in combination with one or moreadditional anti-cancer agents, as described herein are administered viacontinuous infusion for at least 24, 48, 72, 96, or 120 hours. Incertain embodiments, the continuous administration can be repeated atleast once a week, once every two weeks and/or once a month. In otherembodiments, the TCN, TCN-P, TCN-PM and/or related compounds incombination with one or more additional anti-cancer agents, as describedherein can be administered at least once every three weeks. In furtherembodiments, the compounds can be administered at least once a day forat least 2, 3, 4 or 5 days.

In further embodiments, the TCN, TCN-P, TCN-PM and/or related compoundsin combination with one or more additional anti-cancer agents asdescribed herein can be administered to patients in an amount that iseffective in causing tumor regression. The administration the TCN,TCN-P, TCN-PM and/or related compounds and at least one additionalanti-cancer agent can provide at least a partial, such as at least 15,20 or 30%, or complete response in vivo in at least 15-20% of thesubjects. In certain embodiments, at least about 2, 5, 10, 15, 20, 30 or50 mg/m² of the TCN, TCN-P, TCN-PM and/or related compounds and at leastabout 0.1, 1, 2, 5, 10, 15, 20, 30 or 50 mg of one or more additionalanti-cancer agents, as described herein can be administered to asubject. The administration of the TCN, TCN-P, TCN-PM and/or relatedcompounds in combination with one or more additional anti-cancer agents,as described herein can be conducted according to any of the therapeuticregimens disclosed herein. In particular embodiments, the dosing regimencan include administering less than 20 mg/m² of the TCN, TCN-P, TCN-PMand/or related compounds in combination with one or more additionalanti-cancer agents, as described herein. In one embodiment, less than 10mg/m² of the TCN, TCN-P, TCN-PM and/or related compounds in combinationwith one or more additional anti-cancer agents, as described herein canbe administered once a week. In further embodiments, dosages of or lessthan 2 mg/m², 5 mg/m², 10 mg/m², and/or 15 mg/m² of the TCN, TCN-P,TCN-PM and/or related compounds and at least about 0.1, 1, 2, 5, 10, 15,20, 30 or 50 mg of one or more additional anti-cancer agents, asdescribed herein can be administered to a subject. In anotherembodiment, less than 10 mg/m² of TCN, TCN-P, TCN-PM and/or relatedcompounds in combination with one or more additional anti-cancer agents,as described herein can be administered to a subject via continuousinfusion for at least five days. In particular embodiments, the TCN,TCN-P, TCN-PM and/or related compounds in combination with one or moreadditional anti-cancer agents, as described herein as disclosed hereincan be used for the treatment of pancreatic, prostate, colorectal and/orovarian cancer.

In another embodiment, the TCN, TCN-P, TCN-PM and/or related compoundsin combination with one or more additional anti-cancer agents, asdescribed herein can be used to prevent and/or treat a carcinoma,sarcoma, lymphoma, leukemia, and/or myeloma. In other embodiments of theinvention, the TCN, TCN-P, TCN-PM and/or related compounds incombination with one or more additional anti-cancer agents, as describedherein can be used to treat solid tumors. In still further embodiments,the TCN, TCN-P, TCN-PM and/or related compounds in combination with oneor more additional anti-cancer agents, as described herein andcompositions disclosed herein can be used for the treatment of a tumoror cancer, such as, but not limited to cancer of the following organs ortissues: breast, prostate, bone, lung, colon, including, but not limitedto colorectal, urinary, bladder, non-Hodgkin lymphoma, melanoma, kidney,renal, pancreas, pharnx, thyroid, stomach, brain, and/or ovaries.

In a particular embodiment, the TCN, TCN-P, TCN-PM and/or relatedcompounds in combination with one or more additional anti-cancer agents,as described herein can be used for the treatment of pancreatic, breast,colorectal and/or ovarian cancer. In further embodiments of theinvention, the TCN, TCN-P, TCN-PM and/or related compounds incombination with one or more additional anti-cancer agents, as describedherein disclosed herein can be used in the treatment ofangiogenesis-related diseases. In certain embodiments, methods areprovided to treat leukemia via continuous infusion of, the TCN, TCN-P,TCN-PM and/or related compounds and one or more taxanes via continuousinfusion for at least 24, 48, 72 or 96 hours. In other embodiments, thecontinuous infusion can be repeated, for example, at least once everytwo, three or four weeks.

In another embodiment, there is provided a method for the treatment ofrumors, cancer, and others disorders associated with an abnormal cellproliferation in a host, the method including administering to the hostan effect amount of the TCN, TCN-P, TCN-PM and/or related compounds incombination with one or more additional anti-cancer agents, as describedherein optionally in combination with a pharmaceutically acceptablecarrier.

In one aspect, the TCN, TCN-P, TCN-PM and/or related compounds incombination with one or more additional anti-cancer agents, as describedherein and compositions can be administered in combination and can formpart of the same composition, or be provided as a separate compositionfor administration at the same time or a different time.

In other embodiments, the TCN, TCN-P, TCN-PM and/or related compounds incombination with one or more additional anti-cancer agents, as describedherein can be used to treat tumors or cancers resistant to one or moredrugs, including the embodiments of tumors or cancers and drugsdisclosed herein. In one embodiment, the TCN, TCN-P, TCN-PM and/orrelated compounds in combination with one or more additional anti-canceragents, as described herein as disclosed herein is administered in aneffective amount for the treatment of a patient with a drug resistanttumor or cancer, for example, multidrug resistant tumors or cancerincluding, but not limited to, those resistant to taxol alone,rapamycin, tamoxifen, cisplatin, and/or gefitinib (iressa).

In certain embodiments, a method is provided including administering toa host in need thereof an effective amount of TCN, TCN-P, TCN-PM and/orrelated compounds in combination with one or more additional anti-canceragents, as described herein, or pharmaceutical composition including aTCN, TCN-P, TCN-PM and/or related compounds and one or more taxanes, inan effective amount for the treatment of tumors, cancer, and othersdisorders associated with an abnormal cell proliferation in a host.

In another embodiment, a method for the treatment of a tumor or canceris provided including an effective amount of a TCN, TCN-P, TCN-PM and/orrelated compounds and one or more additional anti-cancer agentsdisclosed herein, or a salt, isomer, prodrug or ester thereof, to anindividual in need thereof, wherein the cancer is for example,carcinoma, sarcoma, lymphoma, leukemia, or myeloma. The compound, orsalt, isomer, prodrug or ester thereof, is optionally provided in apharmaceutically acceptable composition including the appropriatecarriers, such as water, which is formulated for the desired route ofadministration to an individual in need thereof. Optionally the compoundis administered in combination or alternation with at least oneadditional therapeutic agent for the treatment of rumors or cancer.

Also within the scope of the invention is the use of a TCN, TCN-P,TCN-PM and/or related compounds in combination with one or moreadditional anti-cancer agents, as described herein or a salt, prodrug orester thereof in the treatment of a tumor or cancer, optionally in apharmaceutically acceptable carrier; and the use of a TCN, TCN-P, TCN-PMand/or related compounds in combination with one or more additionalanti-cancer agents, as described herein or a salt, prodrug or esterthereof in the manufacture of a medicament for the treatment of canceror tumor, optionally in a pharmaceutically acceptable carrier.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the identification of API-2 (triciribine) as acandidate of Akt inhibitor from the NCI Diversity Set. FIG. 1Aillustrates the chemical structure of API-2 (triciribine). FIG. 1Bdemonstrates that API-2 inhibits phosphorylation levels of AKT2 inAKT2-transformed NIH3T3 cells. Wile type AKT2-transformed NIH3T3 cellswere treated with API-2 (1 μM) for indicated times and subjected toimmunoblotting analysis with anti-phospho-Akt-T308 and -S473 antibodies(top and middle panels). The bottom panel shows expression of totalAKT2. In FIG. 1C, it is shown that API-2 inhibits three isoforms of Akt.HEK293 cells were transfected with HA-Akt1, -AKT2 and -AKT3 and treatedwith API-2 (1 μM) or wortmannin (15 μM) prior to EGF stimulation, thecells were lysed and immunoprecipitated with anti-HA antibody. Theimmunoprecipitates were subjected to in vitro kinase assay (top) andimmunoblotting analysis with anti-phospho-Akt-T308 (bottom) antibody.Middle panel shows expression of transfected Akt1, AKT2 and AKT3. FIG.1D illustrates that API-2 did not inhibit Akt in vitro. In vitro kinaseassay of constitutively active AKT2 recombinant protein in a kinasebuffer containing 1 μM API-2 (lane 3).

FIG. 2 demonstrates that API-2 does not inhibit PI3K, PDK1 and theclosely related members of AGC kinase family. FIG. 2A demonstrates an invitro PI3K kinase assay. HEK293 cells were serum-starved and treatedwith API-2 (1 μM) or Wortmannin (15 μM) for 30 minutes prior to EGFstimulation. Cells were lysed and immunoprecipitated with anti-p110ααantibody. The immunoprecipitates were subjected to in vitro kinase assayusing P1-4-P as substrate. FIG. 2B illustrates the effect of API-2 on invitro PDK1 activation (top panel), closed circles show inhibition byAPI-2. Open circles show inhibition by the positive controlstaurosporine, which is a potent PDK1 inhibitor (IC50=5 nM). Bottompanels are immunoblotting analysis of HEK293 cells that were transfectedwith Myc-PDK1 and treated with wortmannin or API-2 prior to EGFstimulation. The immunoblots were detected with indicated antibodies.FIG. 2C illustrates an immunoblot analysis of phosphorylation levels ofPKCα with anti-phospho-PKCα T638 (top) and total PKCα (bottom)antibodies following treatment with API-2 or a nonselective PKCinhibitor Ro31-8220. FIG. 2D shows an in vitro SGK kinase assay. HEK293cells were transfected with HA-SGK and treated with API-2 or wortmanninprior to EGF stimulation. In vitro kinase was performed with HA-SGKimmunoprecipitates using MBP as substrate (top). Bottom panel shows theexpression of transfected HA-SGK. FIG. 2E illustrates the results of aPKA kinase assay. Immuno-purified PKA was incubated in ADB buffer(Upstate Biotechnology Inc) containing indicated inhibitors (API-2 orPKA1) and substrate Kemptide. The kinase activity was quantified. InFIG. 2F, a western blot is shown. OVCAR3 cells were treated with API-2for indicated times. Cell lysates were immunoblotted with indicatedanti-phospho-antibodies (panels 1-4) and anti-actin antibody (bottom).

FIG. 3 demonstrates that API-2 inhibits Akt activity and cell growth andinduces apoptosis in human cancer cells with elevated Akt. FIG. 3A is awestern blot, following treatment with API-2, phosphorylation levels ofAkt were detected with anti-phospho-Akt-T308 antibody in indicated humancancer cell lines. The blots were reprobed with anti-total Akt antibody(bottom panels). In FIG. 3B, a cell proliferation assay is shown. Celllines as indicated in the figure were treated with different doses ofAPI-2 for 24 h and 48 h and then analyzed with CellTiter 96 CellProliferation Assay kit (Promega). FIG. 3C provides an apoptosisanalysis. Cells were treated with API-2 and stained with annexin V andPI and analyzed by FACScan.

FIG. 4 shows that API-2 inhibits downstream targets of Akt and exhibitsanti-tumor activity in cancer cell lines with elevated Akt in mousexenograft. In FIG. 4A, it is demonstrated that API-2 inhibits Aktphosphorylation of tuberin, Bad, AFX and GSK-3β. Following treatmentwith API-2, OVAR3 cells were lysed and immunoblotted with indicatedantibodies. FIG. 4B shows that API-2 inhibits tumor growth. Tumor cellswere subcutaneously injected into nude mice with low level of Akt cellson left side and elevated level of Akt cells on right side. When thetumors reached an average size of about 100-150 mm³, animals weretreated with either vehicle or 1 mg/kg/day API-2. Each measurementrepresents an average of 10 tumors. FIG. 4C illustrates a representationof the mice with OVCAR3 (right) and OVCAR5 (left) xenograft treated withAPI-2 or vehicle (control). FIG. 4D shows examples of tumor size(bottom) and weight (top) at the end of experiment. In FIG. 4E,immunoblot analysis of tumor lysates was performed withanti-phospho-Akt-S473 (top) and anti-AKT2 (bottom) antibodies inOVCAR-3-derived tumors that were treated (T3 and T4) and untreated (T1and T2) with API-2.

FIG. 5 shows that API-2 (triciribine) inhibits Akt kinase activity invitro. In vitro kinase assay was performed with recombinant of PDK1 andAkt in a kinase buffer containing phosphatidylinositol-3,4,5-P3 (PIP3),API-2 and histone H2B as substrate. After incubation of 30 min, thereactions were separated by SDS-PAGE and exposed in a film.

FIG. 6 provides the mRNA and amino acid sequence of human Akt 1,restriction enzyme sites are also noted.

FIG. 7 provides the mRNA and amino acid sequence of human Akt2restriction enzyme sites are also noted.

FIG. 8 provides the mRNA and amino acid sequence of human Akt3restriction enzyme sites are also noted.

FIG. 9 shows the synergistic effect of a combination of triciribine andTaxol on different cancer cell lines.

FIG. 10 shows growth inhibition by the combination of trastuzumab andAkt/mTOR pathway inhibitors. PTEN anti-sense or non-specificoligonucleotide transfected BT474.m1 cells were treated with inhibitorsof the Akt/mTOR pathway alone or in combination with trastuzumab and therelative cell growth was assessed. FIG. 10A shows a panel of Akt/mTORinhibitors. Growth inhibition was assessed in PTEN AS transfectedBT474.m1cells. The doses shown are:

Triciribine (TCN) 1μM; RAD001 0.2 nM; QLT0267 10 μM; KP372-1 0.05 μM;4ADPIB 5 μM; Edelfosine 7.5 μM; and trastuzumab (Ttzm) 2 μg/ml. Thestandard deviation (SD) in the percent growth inhibition is indicated.Results shown are the combined data from 2-3experiments with triplicatesof each treatment within each experiment. FIG. 10B shows TCN inhibitscell growth in combination with trastuzumab. BT474ml cells weretransfected with PTEN AS oligonucleotide or non-specific (NS)oligonucleotide, treated with trastuzumab and TCN, alone and incombination, at multiple doses of TCN and assayed for growth inhibition.Trastuzumab was administered at a single concentration. FIG. 10C showsRAD001 inhibits cell growth in combination with trastuzumab. BT474mlcells were transfected with PTEN AS oligonucleotide or non-specific (NS)oligonucleotide, treated with trastuzumab and RAD001, alone and incombination, at multiple doses of RAD001 and assayed for growthinhibition. For FIGS. 10B and 10C: * indicates a significant differencein growth inhibition following combination treatment as compared toeither trastuzumab or TCN/RAD001 alone. P<0.05 was consideredsignificant. Error bars depict the SEM.

FIG. 11 shows the synergistic effects on apoptosis. Twenty-four hoursafter plating, PTEN AS and NS transfected BT474.m1 cells were treated asindicated with trastuzumab (Ttzm), TCN and/or RAD001 at the followingconcentrations: trastuzumab 2 μg/ml; triciribine 2.5 μM; RAD001 0.4 nM.Apo-BrdU Tunel assays were performed to assess apoptosis. The experimentwas performed 3 times and the data shown is the mean apoptosis. Errorbars depict the standard deviation. Trastuzumab+triciribine treatmentsignificantly induced apoptosis (p<0.01) as compared with all othertreatments.

FIG. 12 shows inhibition of Akt and p70S6K activity. To evaluate theeffects of these drugs on the Akt/mTOR pathway, PTEN AS and NSoligonucleotides were transfected int BT474.m1 cells. Two days later,the cells were treated for 2 hours with trastuzumab and triciribine(TCN) (FIG. 12A) or trastuzumab and RAD001 (FIG. 12B). Total celllysates were collected, separated by SDS-PAGE and immunoblotted asindicated. The concentration of trastuzumab was 2 μg/ml, triciribine was2.5 μM and RAD001 was 0.4 nM. The experiments were repeated at leasttwice to insure that results were reproducible.

FIG. 13 shoes combination treatments inhibited tumor growth in a SCIDmice xenograft model. SCID mice received BT474.m1 breast cancer cellxenografts in mammary fat pad. The xenografts grew for 3 weeks togenerate rumors with an average size of 100-150 mm³. PTEN antisenseoligonucleotides, trastuzumab, triciribine (FIG. 13A) and RAD001 (FIG.13B) were administered. The tumors were measured twice weekly withcalipers and tumor size was averaged for each treatment group. Errorbars denote the standard error of the mean. * indicates a significantdifference in growth inhibition following combination treatment ascompared to either trastuzumab (Ttzm), TCN or DMSO alone. P<0.05 wasconsidered significant.

FIG. 14 shows that cisplatin (CDDP) resistant cell lines endogenouslyexpress hyperactive Akt. FIG. 14A shows a graph of cell survival for twoovarian cancer cell lines (A2780S and OV2008) and their cisplatinresistant ocunterparts (A2780CP and C13, respectively) treated withincreasing amounts of cisplatin. The cisplatin resistant variants,A2780CP and C13, demonstrated increased survival in response tocisplatin. FIG. 14B shows that a comparison of cisplatin sensitiveovarian cancer cells, A2780S and OV2008, to their cisplatin resistantcounterparts. The upper panels illustrate hyperactive Akt in thecisplatin resistant cell lines C13 and A2780S. The lower panelsillustrate comparable total amounts of Akt are present in each celltype.

FIG. 15 illustrates that triciribine (TCN) (API-2) overcomes cisplatinresistance. Cisplatin resistant cell lines, A2780S and C13, were treatedwith 0, 5, 10, and 20 μM cisplatin. The white and striped bars representtreatment with cisplatin only. The black and grey bars representtreatment with cisplatin and 10 μM TCN. The co-treatment with TCNdecreased cell survival, indicating TCN overcomes cisplatin resistance.

FIG. 16 illustrates the synergistic effect seen with TCN and cisplatinon the cell survival of C13 ovarian cancer cells. C13 cells were treatedwith 0, 1, 5, 10 and 20 μM TCN, with and without 10 μM cisplatin. Thecombination of cisplatin and TCN synergistically reduced cell survival.

FIG. 17 illustrates that TCN enhances the ability of cisplatin toinhibit the growth of C13 ovarian cancels in nude mice xenografts. Nudemice received C13 xenografts, followed by treatment with vehicle (DMSO),cisplatin alone, TCN alone, or cisplatin and TCN, followed by analysisof tumor progression at 7, 14, 21, or 28 days post-inoculation. The leftpanel (A) shows a graph illustrating that the combination of cisplatinand TCN significantly reduces tumor growth. The right panel (B) depictsrepresentative tumor mass in inoculated mice receiving the representedtreatments.

FIG. 18 shows the rapid activation of Akt pathway by mTOR inhibitors inhuman ovarian cancer cells. The upper panels (A) show western blots ofOV3 cell lysates following the treatment with 1 nM mTOR inhibitorrapamycin for the time indicated. Membranes were probed with antibodiesspecific to phosphorylated isoforms and to the total protein forcomparison of Akt, p70S6K and FKHRL 1. The lower panels (B) illustratewestern blots of MCF7 cell lysates following treatment with the mTORinhibitor RAD001 for the time indicated. Membranes were probed withantibodies specific to phosphorylated isoforms and to the total proteinfor comparison of Akt and p70S6K.

FIG. 19 shows that activation of AKT pathway via mTOR inhibitors isattenuated by TCN. The left panel (A) illustrates the effect of TCN onAkt phosphorylation with rapamycin treatment in OV3 cells. The rightpanel (B) illustrates the same effect of TCN on Akt phosphorylation withRAD001 treatment in MCF7 cells.

FIG. 20 illustrates the enhanced effect on cell growth through thecombination of TCN with RAD001 or rapamycin. The top left panel (A) is agraph illustrating cell number counts of DU-145 cells over six daystreated with control, rapamycin alone, TCN alone, or TCN and rapamycin.The top right panel (B) is a graph illustrating cell number counts ofMCF7 cells over six days treated with control, RAD001 alone, TCN alone,or TCN and RAD001. The bottom panel (C) is a graph illustrating cellnumber counts of OV3 cells over six days treated with control, rapamycinalone, TCN alone, or TCN and rapamycin.

FIG. 21 illustrates the induction of cell survival and chemoresistancethrough Aurora-A activation of Akt. The top panels in FIG. 21A showsuccessfully transfection and expression of Aurora-A in A2780S, A2780CPand OV2008 ovarian cancer cell lines. The second panel in FIG. 21A is agraph illustrating that expression of Aurora-A in A2780S cells increasescell survival and resistance to paclitaxel. FIG. 21B is a graphillustrating that expression of Aurora-A in A2780S cells increases cellsurvival and resistance to cisplatin.

FIG. 22 shows the effect of TCN (API-2) on Aurora-A induced cisplatinresistance. The top graph shows A2780S cells transfected with emptyvector or Aurora-A. The bottom graphs illustrate OV2008 cellstransfected with empty vector or Aurora-A, followed by treatment withcisplatin alone, API-2 alone or API-2 and cisplatin.

FIG. 23 illustrates the effect of Aurora-A on cytochrome c release andAkt activation. FIG. 23A shows immunofluorescence of cytochrome c inA2780S cells transfected with empty vector or Aurora-A treated with orwithout cisplatin. Cisplatin induced release of cytochrome c isinhibited by expression of Aurora-A. FIG. 23B is a western blot showingexpression of Aurora-A increases phosphorylation of Akt in cisplatinsensitive cells. FIG. 23C shows that increased concentrations ofAurora-A increase the phosphorylation and activation of Akt.

FIG. 24 shows a schematic cartoon of the roles API-2, Aurora-A, p53 andAkt play in cell survival.

FIG. 25 shows the effect of the Src family tyrosine kinase inhibitordasatinib (Sprycel®) on Src phosphorylation and Akt phosphorylation in.A375 cells. Inhibition of Src tyrosine kinases increases phosphorylationof Akt.

FIG. 26 shows bortezomib synergistically enhances the effect of API-2(TCN) on the survival myeloma cells. H929 cells (multiple myelomaderived) were treated with 0, 2.5, 5, 10, 20, or 40 nM bortezomib aloneor in the presence of 10 μM API-2, or with 0, 2.5, 5, 10, 20, or 40 μMAPI-2 alone. Cell survival rates were calibrated and graphed (FIG. 26A).U266 cells (multiple myeloma derived) were treated with 0.2.5, 5, 10,20, or 40 μM API-2 alone or in the presence of 10 nM API-2, or with 0,2.5, 5, 10, 20, or 40 nM bortezomib alone. Cell survival rates werecalibrated and graphed (FIG. 26B).

FIG. 27 shows the synergistic effect of bortezomib and API-2 (TCN) onmantle cell lymphoma cells. Jeko-1 cells (mantel cell lymphoma derived)were treated with 0, 2.5, 5, 10, 20, 40, or 80 nM bortezomib, alone orin the presence of 5 μM API-2, or with 0, 1.25, 2.5, 5, 10, 20, or 40 μMAPI-2. Cell survival rates were calibrated and graphed.

5. DETAILED DESCRIPTION OF THE INVENTION

The inventors have determined, contrary to the prior art and experience,how to successfully use TCN, TCN-P, TCN-PM and/or related compounds incombination with one or more taxanes to treat tumors and cancer by oneor a combination of (i) administering TCN, TCN-P, TCN-PM and/or relatedcompounds and one or more taxanes to patients which according to adiagnostic test described below, exhibit enhanced sensitivity to theTCN, TCN-P, TCN-PM and/or related compounds and/or the taxane; (ii)using a described dosage level that minimizes the toxicity of the TCN,TCN-P, TCN-PM and/or related compounds and/or the taxane but yet stillexhibits efficacy; or (iii) using a described dosage regimen thatminimizes the toxicity of the TCN, TCN-P, TCN-PM and/or relatedcompounds and/or the taxane.

The inventors have determined, contrary to the prior art and experience,how to successfully use TCN, TCN-P, TCN-PM and/or related compounds incombination with a molecule that modulates the HER2/neu (erbB2)receptor, for example, trastuzumab or a salt thereof, to treat tumorsand cancer by one or a combination of (i) administering TCN, TCN-P,TCN-PM and/or related compounds and a molecule that modulates theHER2/neu (erbB2) receptor, for example, trastuzumab or a salt thereof,to patients, who according to a diagnostic test described below, exhibitenhanced sensitivity to the TCN, TCN-P, TCN-PM and/or related compoundsand/or the trastuzumab or a salt thereof; (ii) using a described dosagelevel that minimizes the toxicity of the TCN, TCN-P, TCN-PM and/orrelated compounds and/or the trastuzumab or a salt thereof but yet stillexhibits efficacy; or (iii) using a described dosage regimen thatminimizes the toxicity of the TCN, TCN-P, TCN-PM and/or relatedcompounds and/or the trastuzumab or a salt thereof.

The inventors have determined, contrary to the prior art and experience,how to successfully use TCN, TCN-P, TCN-PM and/or related compounds incombination with one or more anthracycline analogs to treat rumors andcancer by one or a combination of (i) administering TCN, TCN-P, TCN-PMand/or related compounds and one or more anthracycline analogs topatients which according to a diagnostic test described below, exhibitenhanced sensitivity to the TCN, TCN-P, TCN-PM and/or related compoundsand/or the anthracycline analogs; (ii) using a described dosage levelthat minimizes the toxicity of the TCN, TCN-P, TCN-PM and/or relatedcompounds and/or the anthracycline analogs but yet still exhibitsefficacy; or (iii) using a described dosage regimen that minimizes thetoxicity of the TCN, TCN-P, TCN-PM and/or related compounds and/or theanthracycline analogs.

The inventors have determined, contrary to the prior art and experience,how to successfully use TCN. TCN-P, TCN-PM and/or related compounds incombination with one or more erlotinib-like compounds to treat tumorsand cancer by one or a combination of (i) TCN, TCN-P, TCN-PM and/orrelated compounds and one or more erlotinib-like compounds to patientswhich according to a diagnostic test described below, exhibit enhancedsensitivity to the TCN, TCN-P, TCN-PM and/or related compounds and/orthe erlotinib-like compounds; (ii) using a described dosage level thatminimizes the toxicity of the TCN, TCN-P, TCN-PM and/or relatedcompounds and/or the erlotinib-like compounds but yet still exhibitsefficacy; or (iii) using a described dosage regimen that minimizes thetoxicity of the TCN, TCN-P, TCN-PM and/or related compounds and/or theerlotinib-like compounds.

The inventors have determined, contrary to the prior art and experience,how to successfully use TCN, TCN-P, TCN-PM and/or related compounds incombination with one or more platinum compounds to treat tumors andcancer by one or a combination of (i) administering TCN, TCN-P, TCN-PMand/or related compounds and one or more platinum compounds only topatients which according to a diagnostic test described below, exhibitenhanced sensitivity to the TCN, TCN-P, TCN-PM and/or related compoundsand/or the one or more platinum compounds; (ii) using a described dosagelevel that minimizes the toxicity of the TCN, TCN-P, TCN-PM and/orrelated compounds and/or the one or more platinum compounds but yetstill exhibits efficacy; or (iii) using a described dosage regimen thatminimizes the toxicity of the TCN, TCN-P, TCN-PM and/or relatedcompounds and/or the one or more platinum compounds.

The inventors have determined, contrary to the prior art and experience,how to successfully use TCN, TCN-P, TCN-PM and/or related compounds incombination with bortezomib and derivatives thereof analogs to treattumors and cancer by one or a combination of (i) administering TCN,TCN-P, TCN-PM and/or related compounds and bortezomib and derivativesthereof analogs only to patients which according to a diagnostic testdescribed below, exhibit enhanced sensitivity to the TCN, TCN-P, TCN-PMand/or related compounds and/or the bortezomib and derivatives thereof;(ii) using a described dosage level that minimizes the toxicity of theTCN, TCN-P, TCN-PM and/or related compounds and/or the bortezomib andderivatives thereof analogs but yet still exhibits efficacy; or (iii)using a described dosage regimen that minimizes the toxicity of the TCN,TCN-P, TCN-PM and/or related compounds and/or the bortezomib andderivatives thereof analogs.

5.1. Definitions

As used herein, the term “compounds of the invention” refers tocompounds of formula I-XXIII, and combinations thereof.

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth, i.e., proliferative disorders. Examples ofsuch proliferative disorders include cancers such as carcinoma,lymphoma, blastoma, sarcoma, and leukemia, as well as other cancersdisclosed herein. More particular examples of such cancers includebreast cancer, prostate cancer, colon cancer, squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, pancreatic cancer, cervical cancer, ovarian cancer, livercancer, e.g., hepatic carcinoma, bladder cancer, colorectal cancer,endometrial carcinoma, kidney cancer, and thyroid cancer.

Other non-limiting examples of cancers are basal cell carcinoma, biliarytract cancer; bone cancer; brain and CNS cancer; choriocarcinoma;connective tissue cancer; esophageal cancer; eye cancer; cancer of thehead and neck; gastric cancer; intra-epithelial neoplasm; larynx cancer;lymphoma including Hodgkin's and Non-Hodgkin's lymphoma; melanoma;myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth,and pharynx); pancreatic cancer; retinoblastoma; rhabdomyosarcoma;rectal cancer; cancer of the respiratory system; sarcoma; skin cancer;stomach cancer; testicular cancer; uterine cancer; cancer of the urinarysystem, as well as other carcinomas and sarcomas.

As used herein, the term “tumor” refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues. For example, a particular cancer may becharacterized by a solid mass tumor. The solid tumor mass, if present,may be a primary tumor mass. A primary tumor mass refers to a growth ofcancer cells in a tissue resulting from the transformation of a normalcell of that tissue. In most cases, the primary tumor mass is identifiedby the presence of a cyst, which can be found through visual orpalpation methods, or by irregularity in shape, texture or weight of thetissue. However, some primary tumors are not palpable and can bedetected only through medical imaging techniques such as X-rays (e.g.,mammography), or by needle aspirations. The use of these lattertechniques is more common in early detection. Molecular and phenotypicanalysis of cancer cells within a tissue will usually confirm if thecancer is endogenous to the tissue or if the lesion is due to metastasisfrom another site.

The term alkyl, as used herein, unless otherwise specified, includes asaturated straight, branched, or cyclic, primary, secondary, or tertiaryhydrocarbon of for example C₁ to C₂₄, and specifically includes methyl,trifluoromethyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl,t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl,cyclohexyl, cyclohexylmedlyl, 3-methylpentyl, 2,2-dimethylbutyl, and2,3-dimethylbutyl. The alkyl is optionally substituted, e.g., with oneor more substituents such as halo (F, Cl, Br or I), (e.g., CF₃,2-Br-ethyl, CH₂F, CH₂Cl, CH₂CF₃ or CF₂CF₃), hydroxyl (e.g. CH₂OH), amino(e.g., CH₂NH₂, CH₂NHCH₃ or CH₂N(CH₃)₂), alkylamino, arylamino, alkoxy,aryloxy, nitro, azido (e.g. CH₂N₃), cyano (e.g. CH₂CN), sulfonic acid,sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected,or protected as necessary, as known to those skilled in the art, forexample, as taught in Greene, et al., Protective Groups in OrganicSynthesis, John Wiley and Sons. Second Edition, 1991, herebyincorporated by reference.

The term lower alkyl, as used herein, and unless otherwise specified,refers to a C₁ to C₄ saturated straight, branched, or if appropriate, acyclic (for example, cyclopropyl) alkyl group, including bothsubstituted and unsubstituted forms.

The term alkylamino or arylamino includes an amino group that has one ortwo alkyl or aryl substituents, respectively.

The term amino acid includes naturally occurring and synthetic α, β, γor δ amino acids, and includes but is not limited to, amino acids foundin proteins, i.e. glycine, alanine, valine, leucine, isoleucine,methionine, phenylalanine, tryptophan, proline, serine, threonine,cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysinc,arginine and histidine. In a preferred embodiment, the amino acid is inthe L-configuration. Alternatively, the amino acid can be a derivativeof alanyl, valinyl, leucinyl, isoleuccinyl, prolinyl, phenylalaninyl,tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl,tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl,argininyl, histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isolcuccinyl,β-prolinyl, β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl,β-scrinyl, β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl,β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl orβ-histidinyl. When the term amino acid is used, it is considered to be aspecific and independent disclosure of each of the esters of a naturalor synthetic amino acid, including but not limited to α, β, γ or δglycine, alanine, valine, leucine, isoleucine, methionine,phenylalanine, tryptophan, proline, serine, threonine, cysteine,tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginineand histidine in the D and L-configurations.

The term “protected” as used herein and unless otherwise definedincludes a group that is added to an oxygen, nitrogen, sulfur orphosphorus atom to prevent its further reaction or for other purposes. Awide variety of oxygen and nitrogen protecting groups are known to thoseskilled in the art of organic synthesis (see Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3^(rd) Ed., John Wiley & Sons, Inc., NewYork, N.Y., 1999).

The term aryl, as used herein, and unless otherwise specified, includesphenyl, biphenyl, or naphthyl, and preferably phenyl. The aryl group isoptionally substituted with one or more moieties such as halo, hydroxyl,amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonicacid, sulfate, phosphonic acid, phosphate, or phosphonate, eitherunprotected, or protected as necessary, as known to those skilled in theart, for example, as taught in Greene, et al., Protective Groups inOrganic Synthesis, John Wiley and Sons, 3^(rd) Ed., 1999.

The term alkaryl or alkylaryl includes an alkyl group with an arylsubstituent. The term aralkyl or arylalkyl includes an aryl group withan alkyl substituent.

The term halo, as used herein, includes chloro, bromo, iodo, and fluoro.

The term acyl includes a carboxylic acid ester in which the non-carbonylmoiety of the ester group is selected from straight, branched, or cyclicalkyl or lower alkyl, alkoxyalkyl including methoxymethyl, aralkylincluding benzyl, aryloxyalkyl such as phenoxymethyl, aryl includingphenyl optionally substituted with halogen, C₁ to C₄ alkyl or C₁ to C₄alkoxy, sulfonate esters such as alkyl or aralkyl sulphonyl includingmethanesulfonyl, the mono, di or triphosphate ester, trityl ormonomethoxytrityl, substituted benzyl, trialkylsilyl (e.g.dimethyl-t-butylsilyl) or diphenylmethylsilyl. Aryl groups in the estersoptimally include a phenyl group. The term “lower acyl” refers to anacyl group in which the non-carbonyl moiety is lower alkyl.

As used herein, the term “substantially free of” or “substantially inthe absence of” with respect to enantiomeric purity, refers to acomposition that includes at least 85% or 90% by weight, preferably 95%to 98% by weight, and even more preferably 99% to 100% by weight, of thedesignated enantiomer. In a preferred embodiment, in the methods andcompounds of this invention, the compounds are substantially free ofother enantiomers.

Similarly, the term “isolated” refers to a compound composition thatincludes at least 85% or 90% by weight, preferably 95% to 98% by weight,and even more preferably 99% to 100% by weight, of the compound, theremainder including other chemical species or enantiomers.

The term “independently” is used herein to indicate that the variable,which is independently applied, varies independently from application toapplication. Thus, in a compound such as R″XYR″, wherein R″ is“independently carbon or nitrogen,” both R″ can be carbon, both R″ canbe nitrogen, or one R″ can be carbon and the other R″ nitrogen.

The term “pharmaceutically acceptable salt or prodrug” is usedthroughout the specification to describe any pharmaceutically acceptableform (such as an ester, phosphate ester, salt of an ester or a relatedgroup) of a compound, which, upon administration to a patient, providesthe compound. Pharmaceutically acceptable salts include those derivedfrom pharmaceutically acceptable inorganic or organic bases and acids.Suitable salts include those derived from alkali metals such aspotassium and sodium, alkaline earth metals such as calcium andmagnesium, among numerous other acids well known in the pharmaceuticalart. Pharmaceutically acceptable prodrugs refer to a compound that ismetabolized, for example hydrolyzed or oxidized, in the host to form thecompound of the present invention. Typical examples of prodrugs includecompounds that have biologically labile protecting groups on afunctional moiety of the active compound. Prodrugs include compoundsthat can be oxidized, reduced, aminated, deaminated, hydroxylated,dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated,acylated, deacylated, phosphorylated, dephosphorylated to produce theactive compound.

The term “pharmaceutically acceptable esters” as used herein, unlessotherwise specified, includes those esters of one or more compounds,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of hosts without undue toxicity, irritation,allergic response and the like, are commensurate with a reasonablebenefit/risk ratio, and are effective for their intended use.

The term “subject” as used herein refers to an animal, preferably amammal, most preferably a human. Mammals can include non-human mammals,including, but not limited to, pigs, sheep, goats, cows (bovine), deer,mules, horses, monkeys and other non-human primates, dogs, cats, rats,mice, rabbits or any other known or disclosed herein.

The term “TCN, TCN-P, TCN-PM and/or related compounds” includescompounds encompassed by the compounds of Formulas I to IV.

5.2. Compounds of the Invention

As used herein and unless otherwise indicated, the term “TCN, TCN-P,TCN-PM and/or related compounds,” “triciribine,” “triciribinephosphate,” and “triciribine phosphate monohydrate” and “triciribine andrelated compounds” refers to compounds encompassed by the followingstructures:

wherein each R₂′, R₃′ and R₅′ are independently hydrogen, optionallysubstituted phosphate or phosphonate (including mono-, di-, ortriphosphate or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); amide, sulfonate ester includingalkyl or arylalkyl; sulfonyl, including methanesulfonyl and benzyl,wherein the phenyl group is optionally substituted with one or moresubstituents as for example as described in the definition of an arylgiven herein; optionally substituted arylsulfonyl; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; or cholesterol;or other pharmaceutically acceptable leaving group that, in vivo,provides a compound wherein R₂′, R₃′ or R₅′ is independently H or mono-,di- or tri-phosphate;

wherein R^(x) and R^(y) are independently hydrogen, optionallysubstituted phosphate; acyl (including lower acyl); amide, alkyl(including lower alkyl); aromatic, polyoxyalkylene such aspolyethyleneglycol, optionally substituted arylsulfonyl; a lipid,including a phospholipid; an amino acid; a carbohydrate; a peptide; orcholesterol; or other pharmaceutically acceptable leaving group. In oneembodiment, the compound is administered as a 5′-phosphoether lipid or a5′-ether lipid.

R₁ and R₂ each are independently H, optionally substituted straightchained, branched or cyclic alkyl (including lower alkyl), alkenyl, oralkynyl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl or heteroaryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonyl,alkylsulfonyl, arylsulfonyl, aralkylsulfonyl.

In one embodiment, R₂′ and R₃′ are hydrogen. In another embodiment, R₂′and R5′ are hydrogen. In yet another embodiment, R₂′, R₃′ and R₅′ arehydrogen. In yet another embodiment, R₂′, R₃′, R₅′, R₁ and R₂ arehydrogen.

In another embodiment, the TCN, TCN-P, TCN-PM and/or related compoundshas the following structure:

-   -   wherein R₃ is H, optionally substituted straight chained,        branched or cyclic alkyl (including lower alkyl), alkenyl, or        alkynyl, NH₂, NHR⁴, N(R⁴)₂, aryl, alkoxyalkyl, aryloxyalkyl, or        substituted aryl; and    -   each R⁴ independently is H, acyl including lower acyl, alkyl        including lower alkyl such as but not limited to methyl, ethyl,        propyl and cyclopropyl, alkenyl, alkynyl, cycloalkyl, alkoxy,        alkoxyalkyl, hydroxyalkyl, or aryl. In a subembodiment, R₃ is a        straight chained C1-11 alkyl, iso-propyl, t-butyl, or phenyl.

In another embodiment, the TCN, TCN-P, TCN-PM and/or related compoundsprovided herein have the following structure:

In another embodiment, the triciribine compounds provided herein havethe following structure:

In another embodiment, the triciribine compounds provided herein havethe following structure:

wherein R₆ is H, alkyl, (including lower alkyl) alkenyl, alkynyl,alkoxyalkyl, hydroxyalkyl, arylalkyl, cycloalkyl, NH₂, NHR⁴, NR⁴R⁴, CF₃,CH₂OH, CH₂F, CH₂Cl, CH—CF₃, C(Y³)₃, C(Y³)₂C(Y²)₃, C(═O)OH, C(═O)OR⁴,C(═O)-alkyl, C(═O)-aryl, C(═O)-alkoxyalkyl, C(═(O)NH₂, C(═O)NHR⁴,C(═O)N(R⁴)₂, where each Y³ is independently H or halo; and

-   -   each R⁴ independently is H, acyl including lower acyl, alkyl        including lower alkyl such as but not limited to methyl, ethyl,        propyl and cyclopropyl, alkenyl, alkynyl, cycloalkyl, alkoxy,        alkoxyalkyl, hydroxyalkyl, or aryl.

In a subembodiment, R₆ is ethyl, CH₂CH₂ OH, or CH₂-phenyl.

In another embodiment, the TCN, TCN-P, TCN-PM and/or related compoundsprovided herein have the following structure:

-   -   wherein R₇ is H, halo, alkyl (including lower alkyl), alkenyl,        alkynyl, alkoxy, alkoxyalkyl, hydroxyalkyl, cycloalkyl, nitro,        cyano, OH, OR⁴, NH₂, NHR⁴, NR⁴R⁴, SH, SR⁴, CF₃, CH₂OH, CH₂F,        CH₂Cl, CH₂CF₃, C(Y³)₃, C(Y³)₂C(Y³)₃, C(═O)OH, C(—O)OR⁴,        C(═O)-alkyl, C(═O)-aryl, C(—O)-alkoxyalkyl, C(═O)NH₂, C(O)NHR⁴,        C(═O)N(R⁴)₂, or N₃, where each Y³ is independently H or halo;        and    -   each R⁴ independently is H, acyl including lower acyl, alkyl        including lower alkyl such as but not limited to methyl, ethyl,        propyl and cyclopropyl, alkenyl, alkynyl, cycloalkyl, alkoxy,        alkoxyalkyl, hydroxyalkyl.

In a subembodiment, R₇ is methyl, ethyl, phenyl, chloro or NH₂.

In another embodiment, the TCN, TCN-P, TCN-PM and/or related compoundsprovided herein have the following structures:

In another embodiment, the TCN, TCN-P, TCN-PM and/or related compoundsprovided herein have the following structure:

As used herein and unless otherwise indicated, the term “taxanes” refersto the following formula:

-   -   wherein    -   R¹⁰ and R¹¹ each are independently H, optionally substituted        straight chained, branched or cyclic alkyl (including lower        alkyl), alkenyl, or alkynyl, aryl, CO-alkyl, CO-alkenyl,        CO-alkynyl, CO-aryl or heteroaryl, CO-alkoxyalkyl,        CO-aryloxyalkyl, CO-substituted aryl, sulfonyl, alkylsulfonyl,        arylsulfonyl, aralkylsulfonyl.

In one embodiment, the taxane compound has the following structure:

-   -   wherein R¹¹ each are independently H, optionally substituted        straight chained, branched or cyclic alkyl (including lower        alkyl), alkenyl, or alkynyl, aryl, CO-alkyl, CO-alkenyl,        CO-alkynyl, CO-aryl or heteroaryl, CO-alkoxyalkyl,        CO-aryloxyalkyl, CO-substituted aryl, sulfonyl, alkylsulfonyl,        arylsulfonyl, aralkylsulfonyl.

In another embodiment, the taxane compound has the following structure:

-   -   wherein R¹¹ each are independently H, optionally substituted        straight chained, branched or cyclic alkyl (including lower        alkyl), alkenyl, or alkynyl, aryl, CO-alkyl, CO-alkenyl,        CO-alkynyl, CO-aryl or heteroaryl, CO-alkoxyalkyl,        CO-aryloxyalkyl, CO-substituted aryl, sulfonyl, alkylsulfonyl,        arylsulfonyl, aralkylsulfonyl.

In another embodiment, the taxane compound has the following structure:

In another embodiment, the taxane compound has the following structure:

As used herein and unless otherwise indicated, the term “a molecule thatmodulates the HER2/neu (erbB2) receptor” refers to compounds which arerecombinant DNA-derived humanized monoclonal antibodies that selectivelybinds with high affinity in a cell-based assay to the extra-cellulardomain of the human epidermal growth factor receptor 2 protein, HER2. Incertain embodiments, the antibody is an IgG₁ kappa that contains humanframework regions with the complementary-determining regions of a murineantibody (4D5) that binds to HER2.

As used herein and unless otherwise indicated, the term “trastuzumab”refers to trastuzumab or a salt thereof. In certain embodiment, the termtrastuzumab refers to the compound with the DrugBank accession numberBTD00098 and the trade name Herceptin, which is a recombinant humanizedmonoclonal antibody directed against the human epidermal growth factorreceptor 2 (HER2). HER2 is overexpressed by many adenocarcinomas,particularly breast adenocarcinomas. Trastuzumab binds to HER2 on thecell surface of a tumor cell, thereby inducing an antibody-dependentcell-mediated cytotoxicity against tumor cells that overexpress HER2.

As used herein and unless otherwise indicated, the term “anthracyclineanalogs” refers to a compound of Formula XVIII or Formula XIX, whereinthe compound of Formula XVIII has the following structure:

-   -   R₁, R₄, R₆, R₇ are each independently hydrogen, hydroxy, alkoxy;    -   R₂ and R₃ are each independently hydrogen, alkyl, alkoxy,        hydroxy, halogen;    -   R₅ is hydrogen, an optionally substituted alkyl chained,        branched or cyclic alkyl, hydroxy, alkoxy;    -   R₆ is CO-alkyl, CO-halogen substituted alkyl, CO-aryl or        heteroaryl.

In other embodiments, the term “anthracycline compounds” refers tocompound encompassed by Formula XIX having the following structure:

-   -   R₁ and R₂ are each independently hydrogen, alkyl, alkoxy,        hydroxy, halogen;    -   R₃ and R₄ are each independently hydrogen, optionally        substituted alkyl chained, branched or cyclic alkyl, optionally        substituted alkyl chained, branched or cyclic alcohol,        optionally substituted alkyl chained, branched or cyclic amine.

In an illustrative embodiment of species encompassed by the term“anthracyclin compounds of Formula XVIII or Formula XIX,” includes, butare limited to, a compound with any structure A-G:

As used herein and unless otherwise indicated, the term “epidermalgrowth factor receptor inhibitor” refers to compounds that target theepidermal growth factor receptor (EGFR) tyrosine kinase, which is highlyexpressed and occasionally mutated in various forms of cancer. Incertain embodiments, the compounds bind in a reversible fashion to theadenosine triphosphate (ATP) binding site of the receptor. In certainembodiments, two members of the EGFR family come together to form ahomodimer. These then use the molecule of ATP to autophosphorylate eachother, which causes a conformational change in their intracellularstructure, exposing a further binding site for binding proteins thatcause a signal cascade to the nucleus. By inhibiting the ATP,autophosphorylation is not possible and the signal is stopped.Illustrative examples of epidermal growth factor receptor inhibitorcompounds include erlotinib-like compounds such as for example,gefitinib and erlotinib.

As used herein and unless otherwise indicated, the term “erlotinib-likecompounds” refers to a compound of Formula XX:

-   -   wherein    -   each R₁ and R₂ is independently hydrogen, independently        optionally substituted alkoxy, optionally substituted amine,        aromatic amine, heteroaromatic amine, optionally substituted        straight chained, branched or cyclic alkyl; and    -   each R₃ and R₄ is independently hydrogen, independently        optionally substituted aromatic amine, heteroaromatic amine, or        cyclic amine. In an illustrative embodiment, the erlotinib-like        compounds include, but are limited to, the following structures:

As used herein and unless otherwise indicated, the term “platinumcompounds” refers to a compound of formula XXI.

-   -   wherein:    -   R₁, R₂, R₃ and R₄ are each independently hydrogen, optionally        substituted amine, aromatic amine, heteroaromatic amine,        optionally substituted straight chained, branched or cyclic        alkyl, aromatic, heteroaromatic or cyclic ring formed among R₁,        R₂, R₃ and R₄.

In an illustrative embodiment, the platinum compounds include, but arelimited to, platinum compounds of structures A-I:

As used herein and unless otherwise indicated, the term “bortezomib andderivatives thereof” refers to a compound of formula XXII:

-   -   wherein    -   R₁, R₂, R₃, R₄, and R₅ each are independently H, optionally        halogenated, substituted straight chained, branched or cyclic        alkyl (including lower alkyl), alkoxyl, alkenyl, or alkynyl,        aryl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl or heteroaryl,        CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonyl,        alkylsulfonyl, arylsulfonyl, aralkylsulfonyl.

In one embodiment, the bortezomib and derivatives thereof have thefollowing structure:

-   -   wherein    -   R₁, R₂, R₃, R₄, and R₅ each are independently H, optionally        halogenated, substituted straight chained, branched or cyclic        alkyl (including lower alkyl), alkoxyl, alkenyl, or alkynyl,        aryl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl or heteroaryl,        CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonyl,        alkylsulfonyl, arylsulfonyl, aralkylsulfonyl.

In another embodiment, the bortezomib and derivatives thereof analogshave the following structure:

Each of the compounds included herein is commercially available or canbe made by syntheses known to those of ordinary skill in the art.

It is to be understood that the compounds disclosed herein may containchiral centers. Such chiral centers may be of either the (R) or (S)configuration, or may be a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, or be stereoisomeric ordiastereomeric mixtures. It is understood that the disclosure of acompound herein encompasses any racemic, optically active, polymorphic,or stereoisomeric form, or mixtures thereof, which preferably possessesthe useful properties described herein, it being well known in the arthow to prepare optically active forms and how to determine activityusing the standard tests described herein, or using other similar testswhich are will known in the art.

Examples of methods that can be used to obtain optical isomers of thecompounds include the following:

(i) physical separation of crystals—a technique whereby macroscopiccrystals of the individual enantiomers are manually separated. Thistechnique can be used if crystals of the separate enantiomers exist,i.e., the material is a conglomerate, and the crystals are visuallydistinct;

(ii) simultaneous crystallization—a technique whereby the individualenantiomers are separately crystallized from a solution of the racemate,possible only if the latter is a conglomerate in the solid state;

(iii) enzymatic resolutions—a technique whereby partial or completeseparation of a racemate by virtue of differing rates of reaction forthe enantiomers with an enzyme

(iv) enzymatic asymmetric synthesis—a synthetic technique whereby atleast one step of the synthesis uses an enzymatic reaction to obtain anenantiomerically pure or enriched synthetic precursor of the desiredenantiomer;

(v) chemical asymmetric synthesis—a synthetic technique whereby thedesired enantiomer is synthesized from an achiral precursor underconditions that produce asymmetry (i.e., chirality) in the product,which may be achieved using chiral catalysts or chiral auxiliaries;

(vi) diastereomer separations—a technique whereby a racemic compound isreacted with an enantiomerically pure reagent (the chiral auxiliary)that converts the individual enantiomers to diastereomers. The resultingdiastereomers are then separated by chromatography or crystallization byvirtue of their now more distinct structural differences and the chiralauxiliary later removed to obtain the desired enantiomer;

(vii) first- and second-order asymmetric transformations—a techniquewhereby diastereomers from the racemate equilibrate to yield apreponderance in solution of the diastereomer from the desiredenantiomer or where preferential crystallization of the diastereomerfrom the desired enantiomer perturbs the equilibrium such thateventually in principle all the material is converted to the crystallinediastereomer from the desired enantiomer. The desired enantiomer is thenreleased from the diastereomer;

(viii) kinetic resolutions—this technique refers to the achievement ofpartial or complete resolution of a racemate (or of a further resolutionof a partially resolved compound) by virtue of unequal reaction rates ofthe enantiomers with a chiral, non-racemic reagent or catalyst underkinetic conditions;

(ix) enantiospecific synthesis from non-racemic precursors—a synthetictechnique whereby the desired enantiomer is obtained from non-chiralstarting materials and where the stereochemical integrity is not or isonly minimally compromised over the course of the synthesis;

(x) chiral liquid chromatography—a technique whereby the enantiomers ofa racemate are separated in a liquid mobile phase by virtue of theirdiffering interactions with a stationary phase. The stationary phase canbe made of chiral material or the mobile phase can contain an additionalchiral material to provoke the differing interactions;

(xi) chiral gas chromatography—a technique whereby the racemate isvolatilized and enantiomers are separated by virtue of their differinginteractions in the gaseous mobile phase with a column containing afixed non-racemic chiral adsorbent phase;

(xii) extraction with chiral solvents—a technique whereby theenantiomers are separated by virtue of preferential dissolution of oneenantiomer into a particular chiral solvent;

(xiii) transport across chiral membranes—a technique whereby a racemateis placed in contact with a thin membrane barrier. The barrier typicallyseparates two miscible fluids, one containing the racemate, and adriving force such as concentration or pressure differential causespreferential transport across the membrane barrier. Separation occurs asa result of the non-racemic chiral nature of the membrane which allowsonly one enantiomer of the racemate to pass through.

In some embodiments, triciribine, triciribine phosphate (TCN-P),triciribine 5′-phosphate (TCN-P), or the DMF adduct of triciribine(TCN-DMF) are provided. TCN can be synthesized by any technique known toone skilled in the art, for example, as described in TetrahedronLetters, 1971. 49: p. 4757-4760. TCN-P can be prepared by any techniqueknown to one skilled in the art, for example, as described in U.S. Pat.No. 4,123,524. The synthesis of TCN-DMF is described, for example, inINSERM, 1978. 81: p. 37-82. Other compounds related to TCN as describedherein can be synthesized, for example, according to the methodsdisclosed in Gudmundsson K. S. et al., Nucleosides Nucleotides NucleicAcids, 2001. 20(10-11): p. 1823-1830; Porcari A. R. et al., J Med Chem,2000. 43(12): p. 2457-2463; Porcari A. R. et al., NucleosidesNucleotides, 1999. 18(11-12): p. 2475-2497; Porcari A. R. et al., J MedChem, 2000. 43(12): p. 2438-2448; Porcari A. R. et al., NucleosidesNucleotides Nucleic Acids, 2003. 22(12): p. 2171-2193; Porcari A. R. etal., Nucleosides Nucleotides Nucleic Acids, 2004. 23(1-2): p. 31-39;Schweinsberg P. D. et al., Biochem Pharmacol, 1981. 30(18): p.2521-2526; Smith K. L. et al., Bioorg Med Chem Lett, 2004. 14(13): p.3517-3520; Townsend L. B. et al., Nucleic Acids Symp Ser, 1986.1986(17): p. 41-44; and/or Wotring L. L. et al., Cancer Treat Rep, 1986.70(4): p. 491-7, each of which is incorporated herein by reference.

5.3. Pharmaceutically Acceptable Salts, Hydrates and Prodrugs

In cases where compounds are sufficiently basic or acidic to form stablenontoxic acid or base salts, administration of the compound as apharmaceutically acceptable salt may be appropriate. Pharmaceuticallyacceptable salts include those derived from pharmaceutically acceptableinorganic or organic bases and acids. Suitable salts include thosederived from alkali metals such as potassium and sodium, alkaline earthmetals such as calcium and magnesium, among numerous other acids wellknown in the pharmaceutical art. In particular, examples ofpharmaceutically acceptable salts are organic acid addition salts formedwith acids, which form a physiological acceptable anion, for example,tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate.Suitable inorganic salts may also be formed, including, sulfate,nitrate, bicarbonate, and carbonate salts.

In another embodiment, the TCN, TCN-P, TCN-PM and/or related compoundsis a solvate, for example, triciribine phosphate monohydrate (TCN-PM).

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

Any of the nucleotides described herein can be administered as anucleotide prodrug to increase the activity, bioavailability, stabilityor otherwise alter the properties of the nucleoside. A number ofnucleotide prodrug ligands are known. In general, alkylation, acylationor other lipophilic modification of the mono, di or triphosphate of thenucleoside will increase the stability of the nucleotide. Examples ofsubstituent groups that can replace one or more hydrogens on thephosphate moiety are alkyl, aryl, steroids, carbohydrates, includingsugars, 1,2-diacylglycerol and alcohols. Many are described in R. Jonesand N. Bischofberger, Antiviral Research, 27 (1995) 1-17. Any of thesecan be used in combination with the disclosed nucleosides to achieve adesired effect.

In one embodiment, the triciribine or a related compound is provided as5′-hydroxyl lipophilic prodrug. Nonlimiting examples of U.S. patentsthat disclose suitable lipophilic substituents that can be covalentlyincorporated into the nucleoside, preferably at the 5′-OH position ofthe nucleoside or lipophilic preparations, include U.S. Pat. Nos.5,149,794 5,194,654 5,223,263 5,256,641 5,411,947 5,463,092 5,543,3895,543,390 5,543,391 and 5,554,728, all of which are incorporated hereinby reference.

Foreign patent applications that disclose lipophilic substituents thatcan be attached to the triciribine or a related compound s of thepresent invention, or lipophilic preparations, include WO 89/02733, WO90/00555, WO 91/16920, WO 91/18914, WO 93/00910, WO 94/26273, WO/15132,EP 0 350 287, EP 93917054.4, and WO 91/19721, each of which isincorporated herein by reference.

Additional nonlimiting examples of derivatives of triciribine or relatedcompounds are those that contain substituents as described in thefollowing publications. These derivatized triciribine or relatedcompounds can be used for the indications described in the text orotherwise as antiviral agents, including as anti-HIV or anti-HBV agents.Ho D. H. W., Cancer Res., 1973 33: p. 2816-2820; Holy A. in Advances inAntiviral Drug Design, Vol. 1, De Clercq (ed.), JAI Press, pp. 179-231;Hong C. I. et al. Biochem Biophys Rs Commun, 1979. 88: p. 1223-1229;Hong C. I. et al., J Med Chem, 1980. 28: p. 171-177; Hostetler K. Y. etal., J Biol Chem, 1990. 266: p. 11714-11717; Hostetler K. Y. et al,Antiviral Res, 1994. 24: p. 59-67; Hostetler K. Y. et al, AntimicrobialAgents Chemother, 1994. 38: p. 2792-2797; Hunston R. N. et al, J MedChem, 1984. 27: p. 440-444; Ji Y. H. et al, J Med Chem, 1990. 33: p.2264-2270; Jones A. S. et al., J Chem Soc Perkin Trans, 1984.1: p.1471-1474, Juodka B. A. and Smart J., Coil Czech Chem Comm, 1974. 39: p.363-968; Kataoka S. et al, Nucleic Acids Res Symn Ser, 1989. 21: p. 1-2;Kataoka S. et al, Heterocycles, 1991. 32: p. 1351-1356; Kinchington D.et al, Antiviral Chem Chemother, 1992. 3: p. 107-112; Kodama K. et al.,Jpn J Cancer Res, 1989. 80: p. 679-685; Korty M. and Engels J.,Naunyn-Schmiedeberg's Arch Pharmacol, 1979. 10: p. 103-111; Kumar A. etal., J Med Chem, 1990. 33: p. 2368-2375; LeBec C. and Huynh-dinh T.,Tetrahedron Lett, 1991. 32: p. 6553-6556; Lichtenstein J. et al., J BiolChem, 1960. 235: p. 457-465; Luethy J. et al., Mitt GegLebensmittelunters Hyg, 1981. 72: p. 131-133 (Chem. Abstr. 95, 127093);McGuigan C. et al., Nucleic Acids Res, 1989. 17: p. 6065-6075; McGuiganC. et al., Antiviral Chem Chemother, 1990. 1: p. 107-113; McGuigan C. etal, Antiviral Chem Chemother, 1990. 1: p. 355-360; McGuigan C. et al.,Antiviral Chem. Chemother. 1990. 1: p. 25-33; McGuigan C. et al.,Antiviral Res, 1991. 15: p. 255-263; McGuigan C. et al., Antiviral Res,1992. 17: p. 311-321; McGuigan C. et al., Antiviral Chem Chemother,1993. 4: p. 97-101; McGuigan C. et al., J Med Chem, 1993. 36: p.1048-1052, each of which is incorporated herein by reference.

Alkyl hydrogen phosphonate derivatives of the anti-HIV agent AZT may beless toxic than the parent nucleoside analogue. Antiviral Chem.Chemother. 5: 271-277; Meyer R. B. et al., Tetrahedron Lett, 1973.269-272; Nagyvary J. et al., Biochem Biophys Res Commun, 1973. 55: p.1072-1077; Namane A. et al., J Med Chem, 1992. 35: p. 3939-3044; NargeotJ. et al., Natl. Acad. Sci. U.S.A., 1983. 80: p. 2395-2399; Nelson K. A.et al., J Am Chem Soc, 1987. 109: p. 4058-4064; Nerbonne J. M. et al.,Nature, 1984. 301: p. 74-76; Neumann J. M. et al., J Am Chem Soc, 11989. 111: p. 4270-4277; Ohno R. et al., Oncology. 1991. 48: p. 451-455.Palomino E. et al., J Med Chem, 1989. 32: p. 622-625; Perkins R. M. etal., Antiviral Res, 1993. 20(Suppl. I): p. 84; Piantadosi C. et al., JMed Chem, 1991. 34: 1408-1414; Pompon A. et al., Antiviral ChemChemother, 1994. 5: p. 91-98; Postemark T., Anu Rev Pharmacol, 1974. 14:p. 23-33; Prisbe E. J. et al., J Med Chem, 1986. 29: p. 671-675; PucchF. et al., Antiviral Res, 1993. 22: p. 155-174; Pugaeva V. P. et al.,Gig Trf Prof Zabol, 1969. 13: p. 47-48 (Chem. Abstr. 72, 212); Robins R.K., Pharm Res, 1984. 11-18; Rosowsky A. et al., J Med Chem, 1982. 25: p.171-178; Ross W., Biochem Pharm, 1961. 8: p. 235-240; Ryu E. K. et al.,J Med Chem, 1982. 25: p. 1322-1329; Saffhill R. and Hume W. J., ChemBiol Interact, 1986. 57: p. 347-355; Saneyoshi M. et al., Chem PharmBull, 1980. 28: p. 2915-2923; Sastry J. K. et al., Mol Pharmacol, 1992.41: p. 441-445; Shaw J. P. et al., 9th Annual AAPS Meeting, 1994. SanDiego, Calif. (Abstract). Shuto S. et al., Tetrahedron Lett, 1987. 28:p. 199-202; Shuto S. et al., Chem Pharm Bull, 1988. 36: p. 209-217. Onepreferred phosphate prodrug group is the S-acyl-2-thioethyl group, alsoreferred to as “SATE,” each of which is incorporated herein byreference.

Additional examples of prodrugs that can be used are those described inthe following patents and patent applications: U.S. Pat. Nos. 5,614,548,5,512,671, 5,770,584, 5,962,437, 5,223,263, 5,817,638, 6,252,060,6,448,392, 5,411,947, 5,744,592, 5,484,809, 5,827,831, 5,696,277,6,022,029, 5,780,617, 5,194,654, 5,463,092, 5,744,461, 4,444,766,4,562,179, 4,599,205, 4,493,832, 4,221,732, 5,116,992, 6,429,227,5,149,794, 5,703,063, 5,888,990, 4810,697, 5,512,671, 6,030,960,2004/0259845, 6,670,341, 2004/0161398, 2002/082242, 5,512,671,2002/0082242, and or PCT Publication Nos WO 90/11079, WO 96/39197, andor WO 93/08807, each of which is incorporated herein by reference.

5.4. In Vivo Efficacy/Dosing Regimens

In another aspect of the invention, dosing regimens are provided thatlimit the toxic side effects of TCN, TCN-P, TCN-PM and/or relatedcompounds. In one embodiment, such dosing regimens minimize thefollowing toxic side effects, including, but not limited to,hepatoxicity, thrombocytopenia, hyperglycemia, vomiting, hypocalcemia,anemia, hypoalbunemia, myelosuppression, hypertriglyceridemia,hyperamylasemia, diarrhea, stomachitis and/or fever.

In another embodiment, the administration TCN, TCN-P, TCN-PM and/orrelated compounds and one or more taxanes provides at least a partial orcomplete response in vivo in at least 15-20% of the subjects. Inparticular embodiments, a partial response can be at least 15, 20, 25,30, 35, 40, 50, 55, 60, 65, 70, 75, 80 or 85% regression of the tumor.In other embodiments, this response can be evident in at least 15, 15,20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85 or 90% of thesubjects treated with the therapy. In further embodiments, such responserates can be obtained by any therapeutic regimen disclosed herein.

In other embodiments, methods are provided to treat a subject that hasbeen diagnosed with cancer by administering to the subject. an effectiveamount of TCN, TCN-P, TCN-PM and/or related compounds and one or moretaxanes according to a dosing schedule that includes administering theTCN, TCN-P, TCN-PM and/or related compounds and/or the taxane one timeper week for three weeks followed by a one week period wherein the drugis not administered (i.e., via a 28 clay cycle). In other embodiments,such 28 day cycles can be repeated at least 2, 3, 4, or 5 times or untilregression of the tumor is evident.

In further embodiments, a 42 day cycle is provided in which thecompounds disclosed herein can be administered once a week for fourweeks followed by a two week period in which the TCN, TCN-P, TCN-PMand/or related compounds and/or the taxane is not administered. In otherembodiments, such 42 day cycles can be repeated at least 2, 3, 4, or 5times or until regression of the tumor is evident. In a particularembodiment, less than 12, less than 11 or less than 10 mg/m² of TCN,TCN-P, TCN-PM and/or related compounds can be administered according toa 42 day cycle. In other particular embodiments, 2, 3, 4, 5, 6, 7, 8, 9,10 or 11 mg/m² of TCN, TCN-P, TCN-PM and/or related compounds can beadministered according to a 42 day cycle. In another particularembodiment, about 1 to about 50 mg of one or more taxanes isadministered. In a particular embodiment, 1, 5, 10, 15, 20, 25, 30, 35,or 40 mg of one or more taxanes can be administered according to a 42day cycle.

In another embodiment, methods are provided to treat cancer in a subjectby administering to the subject a dosing regimen of 10 mg/m² or less ofTCN, TCN-P, TCN-PM and/or related compounds and less than about 30 mg ofone or more taxanes one time per week. In particular embodiments, 0.5,1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5or 10 mg/m² of TCN, TCN-P, TCN-PM and/or related compounds as disclosedherein can be administered one time per week In another particularembodiment, 1, 5, 10, 15, 20, 25, 30, 35, or 40 mg of one or moretaxanes can be administered one time per week.

In embodiments of the invention, the compounds disclosed herein can beadministered simultaneously as a single bolus dose over a short periodof time, for example, about 5, 10, 15, 20. 30 or 60 minutes. In furtherembodiments, dosing schedules are provided in which the compounds areadministered simultaneously via continuous infusion for at least 24, 48,72, 96, or 120 hours. In certain embodiments, the administration of theTCN, TCN-P, TCN-PM and/or related compounds and/or the taxane viacontinuous or bolus injections can be repeated at a certain frequency atleast: once a week, once every two weeks, once every three weeks, once amonth, once every five weeks, once every six weeks, once every eightweeks, once every ten weeks and/or once every twelve weeks. The type andfrequency of administrations can be combined in any manner disclosedherein to create a dosing cycle. The TCN, TCN-P, TCN-PM and/or relatedcompounds and/or the taxane can be repeatedly administered via a certaindosing cycles, for example as a bolus injection once every two weeks forthree months. The dosing cycles can be administered for at least: one,two three, four five, six, seven, eight, nine, ten, eleven, twelve,eighteen or twenty four months. Alternatively, at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 15 or 20 dosing cycles can be administered to apatient. The TCN, TCN-P, TCN-PM and/or related compounds and/or thetaxane can be administered according to any combination disclosedherein, for example, the TCN, TCN-P, TCN-PM and/or related compoundsand/or the taxane can be administered once a week every three weeks for3 cycles.

In further embodiments, the compounds can be administered separately atleast once a day for at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. Suchadministration can be followed by corresponding periods in which theTCN, TCN-P, TCN-PM and/or related compounds and/or the taxane are notadministered.

The TCN, TCN-P, TCN-PM and/or related compounds and one or more taxanesas disclosed herein can be administered to patients in an amount that iseffective in causing tumor regression. The administration of TCN, TCN-P,TCN-PM and/or related compounds and one or more taxanes can provide atleast a partial, such as at least 15, 20 or 30%, or complete response invivo in at least 15-20% of the subjects. In certain embodiments, atleast 2, 5, 10, 15, 20, 30 or 50 mg/m² of a TCN, TCN-P, TCN-PM and/orrelated compounds disclosed herein can be administered to a subject. Incertain embodiments, at least about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5,5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 12, 15, 17, 20, 25, 30, 35,40, 45, 50, 55, 60, 65. 70, 75, 80, 85, 90, 95, 100, 150, 165, 175, 200,250, 300, or 350 mg/m² of TCN, TCN-P, TCN-PM and/or related compoundsdisclosed herein can be administered to a subject. In certainembodiments, 1, 5, 10, 15, 20, 25, 30, 35, or 40 mg of one or moretaxanes can be administered to a subject.

The administration of the compound can be conducted according to any ofthe therapeutic regimens disclosed herein. In particular embodiments,the dosing regimen includes administering less than about 20 mg/m² ofTCN, TCN-P, TCN-PM and/or related compounds and less than about 30 mg ofone or more taxanes either concurrently, sequentially, or conducted overa period of time. In one embodiment, less than 20 mg/m² of TCN, TCN-P,TCN-PM and/or related compounds or related compounds can be administeredonce a week concurrently with less than about 30 mg of one or moretaxanes. In another embodiment, less than 20 mg/m² of TCN or relatedcompounds can be administered once a week and less than about 30 mg ofone or more taxanes can be administered the following week.

In further embodiments, 2 mg/m², 5 mg/m², 10 mg/m², and/or 15 mg/m² ofTCN, TCN-P, TCN-PM and/or related compounds and less than about 30, 25,20, 15, or 10 mg of one or more taxanes can be administered to asubject. in another embodiment, less than 10 mg/m² of a TCN, TCN-P,TCN-PM and/or related compounds and less than about 30 mg of one or moretaxanes can be administered to a subject via continuous infusion for atleast five days. The present invention provides for any combination ofdosing type, frequency, number of cycles and dosage amount disclosedherein.

In another embodiment, the administration of TCN, TCN-P, TCN-PM and/orrelated compounds and a molecule that modulates the HER2/neu (erbB2)receptor, for example, trastuzumab or a salt thereof, provides at leasta partial or complete response in vivo in at least 15-20% of thesubjects. In particular embodiments, a partial response can be at least15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80 or 85% regression ofthe tumor. In other embodiments, this response can be evident in atleast 15, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85 or 90%of the subjects treated with the therapy. In further embodiments, suchresponse rates can be obtained by any therapeutic regimen disclosedherein.

In other embodiments, methods are provided to treat a subject that hasbeen diagnosed with cancer by administering to the subject an effectiveamount of TCN, TCN-P, TCN-PM and/or related compounds and a moleculethat modulates the HER2/neu (erbB2) receptor, for example, trastuzumabor a salt thereof, according to a dosing schedule that includesadministering the TCN, TCN-P, TCN-PM and/or related compounds and/or amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, one time per week for three weeksfollowed by a one week period wherein the drug is not administered(i.e., via a 28 day cycle). In other embodiments, such 28 day cycles canbe repeated at least 2, 3, 4, or 5 times or until regression of thetumor is evident.

In further embodiments, a 42 day cycle is provided in which thecompounds disclosed herein can be administered once a week for fourweeks followed by a two week period in which the TCN, TCN-P, TCN-PMand/or related compounds and/or a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, is notadministered. In other embodiments, such 42 day cycles can be repeatedat least 2, 3, 4, or 5 times or until regression of the tumor isevident. In a particular embodiment, less than 12, less than 11 or lessthan 10 mg/m² of TCN, TCN-P, TCN-PM and/or related compounds can beadministered according to a 42 day cycle. In other particularembodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 15 mg/m² of TCN, TCN-P,TCN-PM and/or related compounds can be administered according to a 42day cycle. In another particular embodiment, about 1 to about 50 mg of amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, is administered. In a particularembodiment, 1, 5, 10, 15, 20, 25, 30, 35, or 40 mg of a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, can be administered according to a 42 day cycle.

In another embodiment, methods are provided to treat cancer in a subjectby administering to the subject a dosing regimen of 10 mg/m² or less ofTCN, TCN-P, TCN-PM and/or related compounds and less than about 30 mg ofa molecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, one time per week. In particularembodiments, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5 or 10 mg/m² of TCN, TCN-P, TCN-PM and/or relatedcompounds as disclosed herein can be administered one time per week Inanother particular embodiment, 1, 5, 10, 15, 20, 25, 30, 35, or 40 mg ofa molecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, can be administered one time per week.

In embodiments of the invention, the compounds disclosed herein can beadministered simultaneously as a single bolus dose over a short periodof time, for example, about 5, 10, 15, 20, 30 or 60 minutes. In furtherembodiments, dosing schedules are provided in which the compounds areadministered simultaneously via continuous infusion for at least 24, 48,72, 96, or 120 hours. In certain embodiments, the administration of theTCN, TCN-P, TCN-PM and/or related compounds and/or a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, via continuous or bolus injections can be repeated at acertain frequency at least: once a week, once every two weeks, onceevery three weeks, once a month, once every five weeks, once every sixweeks, once every eight weeks, once every ten weeks and/or once everytwelve weeks. The type and frequency of administrations can be combinedin any manner disclosed herein to create a dosing cycle. The TCN, TCN-P,TCN-PM and/or related compounds and/or a molecule that modulates theHER2/neu (erbB2) receptor, for example, trastuzumab or a salt thereof,can be repeatedly administered via a certain dosing cycles, for exampleas a bolus injection once every two weeks for three months. The dosingcycles can be administered for at least: one, two three, four five, six,seven, eight, nine, ten, eleven, twelve, eighteen or twenty four months.Alternatively, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15 or 20dosing cycles can be administered to a patient. The TCN, TCN-P, TCN-PMand/or related compounds and/or a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, can beadministered according to any combination disclosed herein, for example,the TCN, TCN-P, TCN-PM and/or related compounds and/or a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, can be administered once a week every three weeks for 3cycles.

In further embodiments, the compounds can be administered separately atleast once a day for at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. Suchadministration can be followed by corresponding periods in which theTCN, TCN-P, TCN-PM and/or related compounds and/or a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, are not administered.

The TCN, TCN-P, TCN-PM and/or related compounds and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, as disclosed herein can be administered to patients in anamount that is effective in causing tumor regression. The administrationof TCN, TCN-P, TCN-PM and/or related compounds and trastuzumab or a saltthereof can provide at least a partial, such as at least 15, 20 or 30%,or complete response in vivo in at least 15-20% of the subjects. Incertain embodiments, at least 2, 5, 10, 15, 20, 30 or 50 mg/m² of a TCN,TCN-P, TCN-PM and/or related compounds disclosed herein can beadministered to a subject. In certain embodiments, at least about 0.5,1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5,10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 150, 165, 175, 200, 250, 300, or 350 mg/m² of TCN, TCN-P,TCN-PM and/or related compounds disclosed herein can be administered toa subject. In certain embodiments, 1, 5, 10, 15, 20, 25, 30, 35, or 40mg of a molecule that modulates the HER2/neu (erbB2) receptor, forexample, trastuzumab or a salt thereof, can be administered to asubject.

The administration of the compound can be conducted according to any ofthe therapeutic regimens disclosed herein. In particular embodiments,the dosing regimen includes administering less than about 20 mg/m² ofTCN, TCN-P, TCN-PM and/or related compounds and less than about 30 mg ofa molecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, either concurrently, sequentially, orconducted over a period of time. In one embodiment, less than 20 mg/m²of TCN, TCN-P, TCN-PM and/or related compounds can be administered oncea week concurrently with less than about 30 mg of a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof. In another embodiment, less than 20 mg/m² of TCN, TCN-P,TCN-PM and/or related compounds can be administered once a week and lessthan about 30 mg of a molecule that modulates the HER2/neu (erbB2)receptor, for example, trastuzumab or a salt thereof, can beadministered the following week.

In further embodiments, 2 mg/m², 5 mg/m², 10 mg/m², and/or 15 mg/m ofTCN, TCN-P, TCN-PM and/or related compounds and less than about 300,250, 200, 150, or 100 mg of a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, can beadministered to a subject. In another embodiment, less than 10 mg/m² ofa TCN, TCN-P, TCN-PM and/or related compounds and less than about 300 mgof a molecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof can be administered to a subject viacontinuous infusion for at least five days. The present inventionprovides for any combination of dosing type, frequency, number of cyclesand dosage amount disclosed herein.

In another embodiment, the administration of TCN, TCN-P, TCN-PM and/orrelated compounds and one or more anthracycline analogs provides atleast a partial or complete response in vivo in at least 15-20% of thesubjects. In particular embodiments, a partial response can be at least15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80 or 85% regression ofthe tumor. In other embodiments, this response can be evident in atleast 15, 15, 20, 25, 30, 35, 40, 50, 55. 60, 65, 70, 75, 80, 85 or 90%of the subjects treated with the therapy. In further embodiments, suchresponse rates can be obtained by any therapeutic regimen disclosedherein.

In other embodiments, methods are provided to treat a subject that hasbeen diagnosed with cancer by administering to the subject an effectiveamount of TCN, TCN-P, TCN-PM and/or related compounds and one or moreanthracycline analogs according to a dosing schedule that includesadministering the TCN, TCN-P, TCN-PM and/or related compounds and/or theanthracycline analogs one time per week for three weeks followed by aone week period wherein the drug is not administered (i.e., via a 28 daycycle). In other embodiments, such 28 day cycles can be repeated atleast 2, 3, 4, or 5 times or until regression of the tumor is evident.

In further embodiments, a 42 day cycle is provided in which thecompounds disclosed herein can be administered once a week for fourweeks followed by a two week period in which the TCN, TCN-P, TCN-PMand/or related compounds and/or anthracycline analogs are notadministered. In other embodiments, such 42 day cycles can be repeatedat least 2, 3, 4, or 5 times or until regression of the tumor isevident. In a particular embodiment, less than 12, less than 11 or lessthan 10 mg/m² of TCN, TCN-P, TCN-PM and/or related compounds can beadministered according to a 42 day cycle. In other particularembodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 mg/m² of TCN, TCN-P,TCN-PM and/or related compounds can be administered according to a 42day cycle. In another particular embodiment, about 1 to about 50 mg ofan anthracycline derivative is administered. In a particular embodiment,1, 5, 10, 15, 20, 25, 30, 35, 40, 50, or 100 mg of an anthracyclinederivative can be administered according to a 42 day cycle.

In another embodiment, methods are provided to treat cancer in a subjectby administering to the subject a dosing regimen of 10 mg/m² or less ofTCN, TCN-P, TCN-PM and/or related compounds and less than about 30 mg ofone or more anthracycline analogs one time per week. In particularembodiments, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5. 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5 or 10 mg/m² of TCN, TCN-P, TCN-PM and/or relatedcompounds as disclosed herein can be administered one time per week Inanother particular embodiment, 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, or100 mg of an anthracycline derivative can be administered one time perweek.

In embodiments of the present invention, the compounds disclosed hereincan be administered simultaneously as a single bolus dose over a shortperiod of time, for example, about 5, 10, 15, 20, 30 or 60 minutes. Infurther embodiments, dosing schedules are provided in which thecompounds are administered simultaneously via continuous infusion for atleast 24, 48, 72, 96, or 120 hours. In certain embodiments, theadministration of the TCN, TCN-P, TCN-PM and/or related compounds and/oranthracycline analogs via continuous or bolus injections can be repeatedat a certain frequency at least: once a week, once every two weeks, onceevery three weeks, once a month, once every five weeks, once every sixweeks, once every eight weeks, once every ten weeks and/or once everytwelve weeks. The type and frequency of administrations can be combinedin any manner disclosed herein to create a dosing cycle. The TCN, TCN-P,TCN-PM and/or related compounds and/or anthracycline analogs can berepeatedly administered via a certain dosing cycles, for example as abolus injection once every two weeks for three months. The dosing cyclescan be administered for at least: one, two three, four five, six, seven,eight, nine, ten, eleven, twelve, eighteen or twenty four months.Alternatively, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15 or 20dosing cycles can be administered to a patient. The TCN, TCN-P, TCN-PMand/or related compounds and/or anthracycline analogs can beadministered according to any combination disclosed herein, for example,the TCN, TCN-P, TCN-PM and/or related compounds and/or anthracyclineanalogs can be administered once a week every three weeks for 3 cycles.

In further embodiments, the compounds can be administered separately atleast once a day for at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. Suchadministration can be followed by corresponding periods in which theTCN, TCN-P, TCN-PM and/or related compounds and/or anthracycline analogsare not administered.

The TCN, TCN-P, TCN-PM and/or related compounds and one or moreanthracycline analogs as disclosed herein can be administered topatients in an amount that is effective in causing tumor regression. Theadministration of TCN, TCN-P, TCN-PM and/or related compounds and one ormore anthracycline analogs can provide at least a partial, such as atleast 15, 20 or 30%, or complete response in vivo in at least 15-20% ofthe subjects. In certain embodiments, at least 2, 5, 10, 15, 20, 30 or50 mg/m² of a TCN, TCN-P, TCN-PM and/or related compounds disclosedherein can be administered to a subject. In certain embodiments, atleast about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,8, 8.5, 9, 9.5, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 150, 165, 175, 200, 250, 300, or 350 mg/m²of TCN, TCN-P, TCN-PM and/or related compounds disclosed herein can beadministered to a subject. In certain embodiments, 1, 5, 10, 15, 20, 25,30, 35, 40, 50, or 100 mg of an anthracycline derivative can beadministered to a subject.

The administration of the compound can be conducted according to any ofthe therapeutic regimens disclosed herein. In particular embodiments,the dosing regimen includes administering less than about 20 mg/m² ofTCN, TCN-P, TCN-PM and/or related compounds and less than about 30 mg ofan anthracycline derivative either concurrently, sequentially, orconducted over a period of time. In one embodiment, less than 20 mg/m²of TCN, TCN-P, TCN-PM and/or related compounds can be administered oncea week concurrently with less than about 30 mg of an anthracyclinederivative. In another embodiment, less than 20 mg/m² of TCN, TCN-P,TCN-PM and/or related compounds can be administered once a week and lessthan about 30 mg of an anthracycline derivative can be administered thefollowing week.

In further embodiments, 2 mg/m², 5 mg/m², 10 mg/m², and/or 15 mg/m² ofTCN, TCN-P, TCN-PM and/or related compounds and less than about 30, 25,20, 15, or 10 mg of an anthracycline derivative can be administered to asubject. In another embodiment, less than 10 mg/m² of a TCN, TCN-P,TCN-PM and/or related compounds and less than about 30 mg of ananthracycline derivative can be administered to a subject via continuousinfusion for at least five days. The present invention provides for anycombination of dosing type, frequency, number of cycles and dosageamount disclosed herein.

In another embodiment, the administration of TCN, TCN-P, TCN-PM and/orrelated compounds and an erlotinib-like compound, for example,gefitinib, erlotinib or a salt thereof, provides at least a partial orcomplete response in vivo in at least 15-20% of the subjects. Inparticular embodiments, a partial response can be at least 15, 20, 25,30, 35, 40, 50, 55, 60, 65, 70, 75, 80 or 85% regression of the tumor.In other embodiments, this response can be evident in at least 15, 15,20, 25, 30. 35, 40, 50, 55, 60, 65, 70, 75, 80, 85 or 90% of thesubjects treated with the therapy. In further embodiments, such responserates can be obtained by any therapeutic regimen disclosed herein.

In other embodiments, methods are provided to treat a subject that hasbeen diagnosed with cancer by administering to the subject an effectiveamount of TCN, TCN-P, TCN-PM and/or related compounds and anerlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof, according to a dosing schedule that includes administering theTCN, TCN-P, TCN-PM and/or related compounds and/an erlotinib-likecompound, for example, gefitinib, erlotinib or a salt thereof, one timeper week for three weeks followed by a one week period wherein the drugis not administered (i.e., via a 28 day cycle). In other embodiments,such 28 day cycles can be repeated at least 2, 3, 4, or 5 times or untilregression of the tumor is evident.

In further embodiments, a 42 day cycle is provided in which thecompounds disclosed herein can be administered once a week for fourweeks followed by a two week period in which the TCN, TCN-P, TCN-PMand/or related compounds and/or an erlotinib-like compound, for example,gefitinib, erlotinib or a salt thereof, is not administered. In otherembodiments, such 42 day cycles can be repeated at least 2, 3, 4, or 5times or until regression of the tumor is evident. In a particularembodiment, less than 12, less than 11 or less than 10 mg/m² of TCN,TCN-P, TCN-PM and/or related compounds can be administered according toa 42 day cycle. In other particular embodiments, 2, 3, 4, 5, 6, 7, 8, 9,10 or 15 mg/m² of TCN, TCN-P, TCN-PM and/or related compounds can beadministered according to a 42 day cycle. In another particularembodiment, about 1 to about 50 mg of an erlotinib-like compound, forexample, gefitinib, erlotinib or a salt thereof is administered. In aparticular embodiment, 1, 5, 10, 15, 20, 25, 30, 35, or 40 mg of anerlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof, can be administered according to a 42 day cycle.

In another embodiment, methods are provided to treat cancer in a subjectby administering to the subject a dosing regimen of 10 mg/m² or less ofTCN, TCN-P, TCN-PM and/or related compounds and less than about 30 mg ofan erlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof, one time per week. In particular embodiments, 0.5, 1. 1.5, 2,2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg/m²of TCN, TCN-P, TCN-PM and/or related compounds as disclosed herein canbe administered one time per week in another particular embodiment, 1,5, 10, 15, 20, 25, 30, 35, or 40 mg of an erlotinib-like compound, forexample, gefitinib, erlotinib or a salt thereof, can be administered onetime per week.

In embodiments of the present invention, the compounds disclosed hereincan be administered simultaneously as a single bolus dose over a shortperiod of time, for example, about 5, 10, 15, 20, 30 or 60 minutes. Infurther embodiments, dosing schedules are provided in which thecompounds are administered simultaneously via continuous infusion for atleast 24, 48, 72, 96, or 120 hours. In certain embodiments, theadministration of the TCN, TCN-P, TCN-PM and/or related compounds and/oran erlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof, via continuous or bolus injections can be repeated at a certainfrequency at least: once a week, once every two weeks, once every threeweeks, once a month, once every five weeks, once every six weeks, onceevery eight weeks, once every ten weeks and/or once every twelve weeks.The type and frequency of administrations can be combined in any mannerdisclosed herein to create a dosing cycle. The TCN, TCN-P, TCN-PM and/orrelated compounds and/or an erlotinib-like compound, for example,gefitinib, erlotinib or a salt thereof, can be repeatedly administeredvia a certain dosing cycles, for example as a bolus injection once everytwo weeks for three months. The dosing cycles can be administered for atleast: one, two three, four five, six, seven, eight, nine, ten, eleven,twelve, eighteen or twenty four months. Alternatively, at least 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 15 or 20 dosing cycles can be administered toa patient. The TCN, TCN-P, TCN-PM and/or related compounds and/or anerlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof, can be administered according to any combination disclosedherein, for example, the TCN, TCN-P, TCN-PM and/or related compoundsand/or an erlotinib-like compound, for example, gefitinib, erlotinib ora salt thereof, can be administered once a week every three weeks for 3cycles.

In further embodiments, the compounds can be administered separately atleast once a day for at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. Suchadministration can be followed by corresponding periods in which theTCN, TCN-P, TCN-PM and/or related compounds and/or an erlotinib-likecompound, for example, gefitinib, erlotinib or a salt thereof, are notadministered

The TCN, TCN-P, TCN-PM and/or related compounds and an erlotinib-likecompound, for example, gefitinib, erlotinib or a salt thereof, asdisclosed herein can be administered to patients in an amount that iseffective in causing tumor regression. The administration of TCN, TCN-P,TCN-PM and/or related compounds and an erlotinib-like compound, forexample, gefitinib, erlotinib or a salt thereof can provide at least apartial, such as at least 15, 20 or 30%, or complete response in vivo inat least 15-20% of the subjects. In certain embodiments, at least 2, 5,10, 15, 20, 30 or 50 mg/m² of a TCN, TCN-P, TCN-PM and/or relatedcompounds disclosed herein can be administered to a subject. In certainembodiments, at least about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5,6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 165, 175, 200, 250,300, or 350 mg/m² of TC TCN, TCN-P, TCN-PM and/or related compoundsdisclosed herein can be administered to a subject. In certainembodiments, 1, 5, 10, 15, 20, 25, 30, 35, or 40 mg of an erlotinib-likecompound, for example, gefitinib, erlotinib or a salt thereof, can beadministered to a subject.

The administration of the compound can be conducted according to any ofthe therapeutic regimens disclosed herein. In particular embodiments,the dosing regimen includes administering less than about 20 mg/m² ofTCN, TCN-P, TCN-PM and/or related compounds and less than about 30 mg ofan erlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof, either concurrently, sequentially, or conducted over a periodof time. In one embodiment, less than 20 mg/m² of TCN, TCN-P, TCN-PMand/or related compounds can be administered once a week concurrentlywith less than about 30 mg of an erlotinib-like compound, for example,gefitinib, erlotinib or a salt thereof. In another embodiment, less than20 mg/m² of TCN, TCN-P, TCN-PM and/or related compounds can beadministered once a week and less than about 30 mg of an erlotinib-likecompound, for example, gefitinib, erlotinib or a salt thereof can beadministered the following week.

In further embodiments, 2 mg/m², 5 mg/nm, 10 mg/m², and/or 15 mg/m² ofTCN or a related compound and less than about 300, 250, 200, 150, or 100mg an erlotinib-like compound, for example, gefitinib, erlotinib or asalt thereof can be administered to a subject. In another embodiment,less than 10 mg/m² of a TCN, TCN-P, TCN-PM and/or related compounds andless than about 300 mg of an erlotinib-like compound, for example,gefitinib, erlotinib or a salt thereof can be administered to a subjectvia continuous infusion for at least five days. The present inventionprovides for any combination of dosing type, frequency, number of cyclesand dosage amount disclosed herein.

In another embodiment, the administration of TCN, TCN-P, TCN-PM and/orrelated compounds and one or more platinum compounds provides at least apartial or complete response in vivo in at least 15-20% of the subjects.In particular embodiments, a partial response can be at least 15, 20,25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80 or 85% regression of therumor. In other embodiments, this response can be evident in at least15, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85 or 90% of thesubjects treated with the therapy. In further embodiments, such responserates can be obtained by any therapeutic regimen disclosed herein.

In other embodiments, methods are provided to treat a subject that hasbeen diagnosed with cancer by administering to the subject an effectiveamount of TCN, TCN-P, TCN-PM or a related compound and one or moreplatinum compounds according to a dosing schedule that includesadministering the TCN, TCN-P, TCN-PM and/or related compounds and/or theone or more platinum compounds one time per week for three weeksfollowed by a one week period wherein the drug is not administered(i.e., via a 28 day cycle). In other embodiments, such 28 day cycles canbe repeated at least 2, 3, 4, or 5 times or until regression of thetumor is evident.

In further embodiments, a 42 day cycle is provided in which thecompounds disclosed herein can be administered once a week for fourweeks followed by a two week period in which the TCN, TCN-P, TCN-PMand/or related compounds and/or one or more platinum compounds are notadministered. In other embodiments, such 42 day cycles can be repeatedat least 2, 3, 4, or 5 times or until regression of the tumor isevident. In a particular embodiment, less than 12, less than 11 or lessthan 10 mg/m² of TCN, TCN-P, TCN-PM or a related compound can beadministered according to a 42 day cycle. In other particularembodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 mg/m² of TCN, TCN-P, TCN-PMor a related compound can be administered according to a 42 day cycle.In another particular embodiment, about 1 to about 50 mg of a platinumcompound is administered. In a particular embodiment, 1, 5, 10, 15, 20,25, 30. 35, 40, 50, 60, 70, 80, 90 or 100 mg/kg of a platinum compoundcan be administered according to a 42 day cycle.

In another embodiment, methods are provided to treat cancer in a subjectby administering to the subject a dosing regimen of 10 mg/m² or less ofTCN, TCN-P, TCN-PM and/or related compounds and less than about 30 mg ofone or more platinum compounds one time per week. In particularembodiments, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5 or 10 mg/m2 of TCN, TCN-P, TCN-PM and/or relatedcompounds as disclosed herein can be administered one time per week Inanother particular embodiment, 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60,70, 80, 90, or 100 mg/kg of a platinum compound can be administered onetime per week.

In embodiments of the present invention, the compounds disclosed hereincan be administered simultaneously as a single bolus dose over a shortperiod of time, for example, about 5, 10, 15, 20, 30 or 60 minutes. Infurther embodiments, dosing schedules are provided in which thecompounds are administered simultaneously via continuous infusion for atleast 24, 48, 72, 96, or 120 hours. In certain embodiments, theadministration of the TCN, TCN-P, TCN-PM and/or related compounds and/orone or more platinum compounds via continuous or bolus injections can berepeated at a certain frequency at least: once a week, once every twoweeks, once every three weeks, once a month, once every five weeks, onceevery six weeks, once every eight weeks, once every ten weeks and/oronce every twelve weeks. The type and frequency of administrations canbe combined in any manner disclosed herein to create a dosing cycle. TheTCN, TCN-P, TCN-PM and/or related compounds and/or one or more platinumcompounds can be repeatedly administered via a certain dosing cycles,for example as a bolus injection once every two weeks for three months.The dosing cycles can be administered for at least: one, two three, fourfive, six, seven, eight, nine, ten, eleven, twelve, eighteen or twentyfour months. Alternatively, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,15 or 20 dosing cycles can be administered to a patient. The TCN, TCN-P,TCN-PM and/or related compounds and/or one or more platinum compoundscan be administered according to any combination disclosed herein, forexample, the TCN, TCN-P, TCN-PM and/or related compounds and/or one ormore platinum compounds can be administered once a week every threeweeks for 3 cycles.

In further embodiments, the compounds can be administered separately atleast once a day for at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. Suchadministration can be followed by corresponding periods in which theTCN, TCN-P, TCN-PM and/or related compounds and/or one or more platinumcompounds are not administered.

The TCN, TCN-P, TCM-PM and related compounds and one or more platinumcompounds as disclosed herein can be administered to patients in anamount that is effective in causing tumor regression. The administrationof TCN, TCN-P, TCN-PM and/or related compounds and one or more platinumcompounds can provide at least a partial, such as at least 15, 20 or30%, or complete response in vivo in at least 15-20% of the subjects. Incertain embodiments, at least 2, 5, 10, 15, 20, 30 or 50 mg/m² of a TCN,TCN-P, TCN-PM and/or related compounds disclosed herein can beadministered to a subject. In certain embodiments, at least about 0.5,1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5,10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 150, 165, 175, 200, 250, 300, or 350 mg/m² of TCN, TCN-P,TCN-PM or a related compound disclosed herein can be administered to asubject. In certain embodiments, 10, 20, 50, 100, 150, 200, 250, 300,350, or 400 mg of a platinum compound can be administered to a subject.

The administration of the compound can be conducted according to any ofthe therapeutic regimens disclosed herein. In particular embodiments,the dosing regimen includes administering less than about 20 mg/m² ofTCN, TCN-P, TCN-PM and/or related compounds and less than about 100 mgof a platinum compound either concurrently, sequentially, or conductedover a period of time. In one embodiment, less than 20 mg/m² of TCN orrelated compounds can be administered once a week concurrently with lessthan about 100 mg of a platinum compound. In another embodiment, lessthan 20 mg/m² of TCN or related compounds can be administered once aweek and less than about 100 mg of a platinum compound can beadministered the following week.

In further embodiments, 2 mg/m², 5 mg/m², 10 mg/m², and/or 15 mg/m² ofTCN or a related compound and less than about 30, 25, 20, 50, or 100 mgof a platinum compound can be administered to a subject. In anotherembodiment, less than 10 mg/m² of a TCN, TCN-P, TCN-PM and/or relatedcompounds and less than about 100 mg of a platinum compound can beadministered to a subject via continuous infusion for at least fivedays. The present invention provides for any combination of dosing type,frequency, number of cycles and dosage amount disclosed herein.

In other embodiments, methods are provided to treat a subject that hasbeen diagnosed with cancer by administering to the subject an effectiveamount of TCN, TCN-P, TCN-PM and/or related compounds and bortezomib andderivatives thereof analogs according to a dosing schedule that includesadministering the TCN, TCN-P, TCN-PM and/or related compounds and/or thebortezomib or salt or derivatives thereof one time per week for threeweeks followed by a one week period wherein the drug is not administered(i.e., via a 28 day cycle). In other embodiments, such 28 day cycles canbe repeated at least 2, 3, 4, or 5 times or until regression of thetumor is evident.

In further embodiments, a 42 day cycle is provided in which thecompounds disclosed herein can be administered once a week for fourweeks followed by a two week period in which the TCN, TCN-P, TCN-PMand/or related compounds and/or the bortezomib and derivatives thereofis not administered. In other embodiments, such 42 day cycles can berepeated at least 2, 3, 4, or 5 times or until regression of the tumoris evident. In a particular embodiment, less than 50, less than 25 orless than 10 mg/m² of TCN, TCN-P, TCN-PM or a related compound can beadministered according to a 42 day cycle. In other particularembodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 mg/m² of TCN, TCN-P,TCN-PM or a related compound can be administered according to a 42 daycycle. In another particular embodiment, about 0.1 mg/m² to about 50mg/m² of bortezomib or a derivative thereof is administered. In aparticular embodiment, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, or 40mg/m² of bortezomib or a salt thereof can be administered according to a42 day cycle.

In another embodiment, methods are provided to treat cancer in a subjectby administering to the subject a dosing regimen of 10 mg/m² or less ofTCN, TCN-P, TCN-PM or a related compound and less than about 30 mg ofbortezomib and derivatives thereof analogs one time per week. Inparticular embodiments, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg/m² of TCN, TCN-P, TCN-PM or arelated compound as disclosed herein can be administered one time perweek In another particular embodiment, 0.1, 0.5, 1, 5, 10, 15, 20, 25,30, 35, or 40 mg/m² of bortezomib or a derivative thereof can beadministered one time per week.

In embodiments of the present invention, the compounds disclosed hereincan be administered simultaneously as a single bolus dose over a shortperiod of time, for example, about 5, 10, 15, 20, 30 or 60 minutes. Infurther embodiments, dosing schedules are provided in which thecompounds are administered simultaneously via continuous infusion for atleast 24, 48, 72, 96, or 120 hours. In certain embodiments, theadministration of the TCN, TCN-P, TCN-PM and/or related compounds and/orthe bortezomib and derivatives thereof analogs via continuous or bolusinjections can be repeated at a certain frequency at least: once a week,once every two weeks; once every three weeks, once a month, once everyfive weeks, once every six weeks, once every eight weeks, once every tenweeks and/or once every twelve weeks. The type and frequency ofadministrations can be combined in any manner disclosed herein to createa dosing cycle. The TCN, TCN-P, TCN-PM and/or related compounds and/orthe bortezomib and derivatives thereof analogs can be repeatedlyadministered via a certain dosing cycles, for example as a bolusinjection once every two weeks for three months. The dosing cycles canbe administered for at least: one, two three, four five, six, seven,eight, nine, ten, eleven, twelve, eighteen or twenty four months.Alternatively, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15 or 20dosing cycles can be administered to a patient. The TCN, TCN-P, TCN-PMand/or related compounds and/or the bortezomib and derivatives thereofanalogs can be administered according to any combination disclosedherein, for example, the TCN, TCN-P, TCN-PM and/or related compoundsand/or the bortezomib and derivatives thereof analogs can beadministered once a week every three weeks for 3 cycles.

In further embodiments, the compounds can be administered separately atleast once a day for at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. Suchadministration can be followed by corresponding periods in which theTCN, TCN-P, TCN-PM and/or related compounds and/or the bortezomib andderivatives thereof analogs are not administered.

The TCN, TCN-P, TCN-PM and related compounds and bortezomib andderivatives thereof analogs as disclosed herein can be administered topatients in an amount that is effective in causing tumor regression. Theadministration of TCN, TCN-P, TCN-PM or related compounds and bortezomiband derivatives thereof analogs can provide at least a partial, such asat least 15, 20 or 30%, or complete response in vivo in at least 15-20%of the subjects. In certain embodiments, at least 2, 5, 10, 15, 20, 30or 50 mg/m² of a TCN, TCN-P, TCN-PM and/or related compounds disclosedherein can be administered to a subject. In certain embodiments, atleast about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5. 5.5, 6. 6.5, 7, 7.5,8, 8.5, 9, 9.5, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 150, 165, 175, 200, 250, 300, or 350 mg/m²of TCN, TCN-P, TCN-PM or a related compound disclosed herein can beadministered to a subject. In certain embodiments, 1, 5, 10, 15, 20, 25,30, 35, or 40 mg/m² of bortezomib can be administered to a subject.

The administration of the compound can be conducted according to any ofthe therapeutic regimens disclosed herein. In particular embodiments,the dosing regimen includes administering less than about 20 mg/m² ofTCN, TCN-P, TCN-PM and/or related compounds and less than about 30 mg ofbortezomib either concurrently, sequentially, or conducted over a periodof time. In one embodiment, less than 20 mg/m² of TCN or relatedcompounds can be administered once a week concurrently with less thanabout 10 mg/m² of bortezomib. In another embodiment, less than 20 mg/m²of TCN or related compounds can be administered once a week and lessthan about 30 mg of bortezomib can be administered the following week.

In further embodiments, 2 mg/m², 5 mg/m², 10 mg/m², and/or 15 mg/m² ofTCN or a related compound and less than about 30, 25, 20, 15, 10, 5, 1,0.5, or 0.1 mg/m² of bortezomib or a salt or a derivative thereof can beadministered to a subject. In another embodiment, less than 10 mg/m² ofa TCN, TCN-P, TCN-PM and/or related compounds and less than about 30mg/m² of bortezomib can be administered to a subject via continuousinfusion for at least five days. The present invention provides for anycombination of dosing type, frequency, number of cycles and dosageamount disclosed herein.

5.5. Screening of Patient Populations

In another embodiment of the invention, methods are provided to identifycancers or tumors that are susceptible to the toxic effects oftriciribine (TCN) and related compounds. In one embodiment, methods areprovided to treat a cancer or tumor in a mammal by (i) obtaining abiological sample from the tumor; (ii) determining whether the cancer ortumor overexpresses Akt kinase or hyperactivated and phosphorylated Aktkinase, and (iii) treating the cancer or tumor with triciribine or arelated compound as described herein. In one embodiment, the biologicalsample can be a biopsy. In other embodiments, the biological sample canbe fluid, cells and/or aspirates obtained from the tumor or cancer.

The biological sample can be obtained according to any technique knownto one skilled in the art. In one embodiment, a biopsy can be conductedto obtain the biological sample. A biopsy is a procedure performed toremove tissue or cells from the body for examination. Some biopsies canbe performed in a physician's office, while others need to be done in ahospital setting. In addition, some biopsies require use of ananesthetic to numb the area, while others do not require any sedation.In certain embodiments, an endoscopic biopsy can be performed. This typeof biopsy is performed through a fiberoptic endoscope (a long, thin tubethat has a close-focusing telescope on the end for viewing) through anatural body orifice (i.e., rectum) or a small incision (i.e.,arthroscopy). The endoscope is used to view the organ in question forabnormal or suspicious areas, in order to obtain a small amount oftissue for study. Endoscopic procedures are named for the organ or bodyarea to be visualized and/or treated. The physician can insert theendoscope into the gastrointestinal tract (alimentary tract endoscopy),bladder (cystoscopy), abdominal cavity (laparoscopy), joint cavity(arthroscopy), mid-portion of the chest (mediastinoscopy), or tracheaand bronchial system (laryngoscopy and bronchoscopy).

In another embodiment, a bone marrow biopsy can be performed. This typeof biopsy can be performed either from the sternum (breastbone) or theiliac crest hipbone (the bone area on either side of the pelvis on thelower back area). The skin is cleansed and a local anesthetic is givento numb the area. A long, rigid needle is inserted into the marrow, andcells are aspirated for study; this step is occasionally uncomfortable.A core biopsy (removing a small bone ‘chip’ from the marrow) may followthe aspiration.

In a further embodiment, an excisional or incisional biopsy can beperformed on the mammal. This type of biopsy is often used when a wideror deeper portion of the skin is needed. Using a scalpel (surgicalknife), a full thickness of skin is removed for further examination, andthe wound is sutured (sewed shut with surgical thread). When the entiretumor is removed, it is referred to as an excisional biopsy technique.If only a portion of the tumor is removed, it is referred to as anincisional biopsy technique. Excisional biopsy is often the methodusually preferred, for example, when melanoma (a type of skin cancer) issuspected.

In still further embodiments, a fine needle aspiration (FNA) biopsy canbe used. This type of biopsy involves using a thin needle to remove verysmall pieces from a tumor. Local anesthetic is sometimes used to numbthe area, but the test rarely causes much discomfort and leaves no scar.FNA is not, for example, used for diagnosis of a suspicious mole, butmay be used, for example, to biopsy large lymph nodes near a melanoma tosee if the melanoma has metastasized (spread). A computed tomographyscan (CT or CAT scan) can be used to guide a needle into a tumor in aninternal organ such as the lung or liver.

In other embodiments, punch shave and/or skin biopsies can be conducted.Punch biopsies involve taking a deeper sample of skin with a biopsyinstrument that removes a short cylinder, or “apple core,” of tissue.After a local anesthetic is administered, the instrument is rotated onthe surface of the skin until it cuts through all the layers, includingthe dermis, epidermis. and the most superficial parts of the subcutis(fat). A shave biopsy involves removing the top layers of skin byshaving it off. Shave biopsies are also performed with a localanesthetic. Skin biopsies involve removing a sample of skin forexamination under the microscope to determine if, for example, melanomais present. The biopsy is performed under local anesthesia.

In particular embodiment, methods are provided to determine whether thetumor overexpresses an Akt kinase. Akt kinase overexpression can referto the phosphorylation state of the kinase. Hyperphosphorylation of Aktcan be detected according to the methods described herein. In oneembodiment, a tumor biopsy can be compared to a control tissue. Thecontrol tissue can be a normal tissue from the mammal in which thebiopsy was obtained or a normal tissue from a healthy mammal. Akt kinaseoverexpression or hyperphosphorylation can be determined if the tumorbiopsy contains greater amounts of Akt kinase and/or Akt kinasephosphorylation than the control tissue, such as, for example, at leastapproximately 1.5, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5,4.7, 5, 5.5, 6, 7, 8, 9, or 10-fold greater amounts of Akt kinase thancontained in the control tissue.

In one embodiment, the present invention provides a method to detectaberrant Akt kinase expression in a subject or in a biological samplefrom the subject by contacting cells, cell extracts, serum or othersample from the subjects or said biological sample with animmunointeractive molecule specific for an Akt kinase or antigenicportion thereof and screening for the level of immunointeractivemolecule-Akt kinase complex formation, wherein an elevated presence ofthe complex relative to a normal cell is indicative of an aberrant cellthat expresses or overexpresses Akt. In one example, cells or cellextracts can be screened immunologically for the presence of elevatedlevels of Akt kinase.

In an alternative embodiment, the aberrant expression of Akt in a cellis detected at the genetic level by screening for the level ofexpression of a gene encoding an Akt kinase wherein an elevated level ofa transcriptional expression product (i.e., mRNA) compared to a normalcell is indicative of an aberrant cell. In certain embodiments,real-time PCR as well as other PCR procedures can be used to determinetranscriptional activity. In one embodiment, mRNA can be obtained fromcells of a subject or from a biological sample from a subject and cDNAoptionally generated. The mRNA or cDNA can then be contacted with agenetic probe capable of hybridizing to and/or amplifying all or part ofa nucleotide sequence encoding Akt kinase or its complementarynucleotide sequence and then the level of the mRNA or cDNA can bedetected wherein the presence of elevated levels of the mRNA or cDNAcompared to normal controls can be assessed.

Yet another embodiment of the present invention contemplates the use ofan antibody, monoclonal or polyclonal, to Akt kinase in a quantitativeor semi-quantitative diagnostic kit to determine relative levels of Aktkinase in suspected cancer cells from a patient, which can include allthe reagents necessary to perform the assay. In one embodiment, a kitutilizing reagents and materials necessary to perform an ELISA assay isprovided. Reagents can include, for example, washing buffer, antibodydilution buffer, blocking buffer, cell staining solution, developingsolution, stop solution, anti-phospho-protein specific antibodies,anti-Pan protein specific antibodies, secondary antibodies, anddistilled water. The kit can also include instructions for use and canoptionally be automated or semi-automated or in a form which iscompatible with automated machine or software. In one embodiment, aphosphor-ser-473 Akt antibody that detects the activated form of AKT(Akt phosphorylated at serine 474) can be utilized as the antibody in adiagnostic kit. See, for example, Yuan et al. (2000) “FrequentActivation of AKT2 and induction of apoptosis by inhibition ofphosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer,”Oncogene 19:2324-2330.

5.6. Akt Kinases

Akt, also named PKB3, represents a subfamily of the serine/threoninekinase. Three members, AKT1, AKT2, and AKT3, have been identified inthis subfamily. Akt is activated by extracellular stimuli in aPI3K-dependent manner (Datta, S. R., et al. Genes Dev. 13: 2905-2927,1999). Full activation of Akt requires phosphorylation of Thr308 in theactivation loop and Ser473 in the C-terminal activation domain. Akt isnegatively regulated by PTEN tumor suppressor. Mutations in PTEN havebeen identified in various tumors, which lead to activation of Aktpathway (Datta, S. R., et al. Genes Dev. 13: 2905-2927. 1999). Inaddition, amplification, overexpression and/or activation of Akt havebeen detected in a number of human malignancies (Datta, S. R., et al.Genes Dev. 13: 2905-2927, 1999, Cheng, J. Q., and Nicosia, S. V. AKTsignal transduction pathway in oncogenesis. In Schwab D, editor.Encyclopedic Reference of Cancer. Berlin Heidelberg and New York:Springer; 2001. pp 35-7). Ectopic expression of Akt, especiallyconstitutively active Akt, induces cell survival and malignanttransformation whereas inhibition of Akt activity stimulates apoptosisin a range of mammalian cells (Datta, S. R., et al. Genes Dev. 13:2905-2927, 1999, Cheng, J. Q., and Nicosia, S. V. AKT signaltransduction pathway in oncogenesis. In Schwab D, editor. EncyclopedicReference of Cancer. Berlin Heidelberg and New York: Springer; 2001. pp35-7, Sun, M., et al. Am. J. Path., 159. 431-437, 2001, Cheng, J. Q., etal. Oncogene, 14: 2793-2801, 1997). Further, activation of Akt has beenshown to associate with tumor invasiveness and chemoresistance (West, K.A., et al. Drug Resist. Updat., 5: 234-248, 2002).

Activation of the Akt pathway plays a pivotal role in malignanttransformation and chemoresistance by inducing cell survival, growth,migration, and angiogenesis. The present invention provides methods todetermine levels of Akt kinase overexpression and/or hyperactivated andphosphorylated Akt kinase.

The Akt kinase can be any known Akt family kinase, or kinase relatedthereto, including, but not limited to Akt 1, Akt 2, Akt 3. The mRNA andamino acid sequences of human Akt1, Akt2, and Akt 3 are illustrated inFIGS. 6 a-c, 7 a-d, and 8 a-c, respectively.

In another embodiment, the compositions of the invention including TCN,TCN-P, TCN-PM and/or related compounds and one or more taxanes killscancer or tumor cells which express Akt.

5.7. Diagnostic Assays

Immunological Assays

In one embodiment, a method is provided for detecting the aberrantexpression of an Akt kinase in a cell in a mammal or in a biologicalsample from the mammal, by contacting cells, cell extracts or serum orother sample from the mammal or biological sample with animmunointeractive molecule specific for an Akt kinase or antigenicportion thereof and screening for the level of immunointeractivemolecule-Akt kinase complex formations and determining whether anelevated presence of the complex relative to a normal cell is present.

The immunointeractive molecule can be a molecule having specificity andbinding affinity for an Akt kinase or its antigenic parts or itshomologs or derivatives thereof. In one embodiment, theimmunointeractive molecule can be an immunoglobulin molecule. In otherembodiments, the immunointeractive molecules can be an antibodyfragments, single chain antibodies, and/or deimmunized moleculesincluding humanized antibodies and T-cell associated antigen-bindingmolecules (TABMs). In one particular embodiment, the antibody can be amonoclonal antibody. In another particular embodiment, the antibody canbe a polyclonal antibody. The immunointeractive molecule can exhibitspecificity for an Akt kinase or more particularly an antigenicdeterminant or epitope on an Akt kinase. An antigenic determinant orepitope on an Akt kinase includes that part of the molecule to which animmune response is directed. The antigenic determinant or epitope can bea B-cell epitope or where appropriate a T-cell epitope. In oneembodiment, the antibody is a phosphor-ser 473 Akt antibody.

One embodiment of the present invention provides a method for diagnosingthe presence of cancer or cancer-like growth in a mammal, in whichaberrant Akt activity is present, by contacting cells or cell extractsfrom the mammal or a biological sample from the subject with an Akikinase-binding effective amount of an antibody having specificity forthe Akt kinase or an antigenic determinant or epitope thereon and thenquantitatively or qualitatively determining the level of an Aktkinase-antibody complex wherein the presence of elevated levels of saidcomplex compared to a normal cell is determined.

Antibodies can be prepared by any of a number of means known to oneskilled in the art. For example, for the detection of human Akt kinase,antibodies can be generally but not necessarily derived from non-humananimals such as primates, livestock animals (e.g. sheep, cows, pigs,goats, horses), laboratory test animals (e.g. mice, rats, guinea pigs,rabbits) and/or companion animals (e.g. dogs, cats). Antibodies may alsobe recombinantly produced in prokaryotic or eukaryotic host cells.Generally, antibody based assays can be conducted in vitro on cell ortissue biopsies. However, if an antibody is suitably deimmunized or, inthe case of human use, humanized, then the antibody can be labeled with,for example, a nuclear tag, administered to a patient and the site ofnuclear label accumulation determined by radiological techniques. TheAkt kinase antibody can be a cancer targeting agent. Accordingly,another embodiment of the present invention provides deimmunized formsof the antibodies for use in cancer imaging in human and non-humanpatients.

In general, for the generation of antibodies to an Akt kinase, theenzyme is required to be extracted from a biological sample whether thisbe from animal including human tissue or from cell culture if producedby recombinant means. The Akt kinase can be separated from thebiological sample by any suitable means. For example, the separation maytake advantage of any one or more of the Akt kinase's surface chargeproperties, size, density, biological activity and its affinity foranother entity (e.g. another protein or chemical compound to which itbinds or otherwise associates). Thus, for example, separation of the Akikinase from the biological fluid can be achieved by any one or more ofultra-centrifugation, ion-exchange chromatography (e.g. anion exchangechromatography, cation exchange chromatography), electrophoresis (e.g.polyacrylamide gel electrophoresis, isoelectric focussing), sizeseparation (e.g., gel filtration, ultra-filtration) andaffinity-mediated separation (e.g. immunoaffinity separation including,but not limited to, magnetic bead separation such as Dynabead(trademark) separation, immunochromatography, immuno-precipitation). Theseparation of Akt kinase from the biological fluid can preserveconformational epitopes present on the kinase and, thus, suitably avoidstechniques that cause denaturation of the enzyme. In a furtherembodiment, the kinase can be separated from the biological fluid usingany one or more of affinity separation, gel filtration and/orultra-filtration.

Immunization and subsequent production of monoclonal antibodies can becarried out using standard protocols known in the art, such as, forexample, described by Kohler and Milstein (Kohler and Milstein, Nature256: 495-499, 1975; Kohler and Milstein, Eur. J. Immunol. 6(7): 511-519,1976), Coligan et al. (“Current Protocols in Immunology, John Wiley &Sons, Inc., 1991-1997) or Toyama et al. (Monoclonal Antibody, ExperimentManual”, published by Kodansha Scientific, 1987). Essentially, an animalis immunized with an Akt kinase-containing biological fluid or fractionthereof or a recombinant form of Akt kinase by standard methods toproduce antibody-producing cells, particularly antibody-producingsomatic cells (e.g. B lymphocytes). These cells can then be removed fromthe immunized animal for immortalization. In certain embodiment, afragment of an Akt kinase can be used to the generate antibodies. Thefragment can be associated with a carrier. The carrier can be anysubstance of typically high molecular weight to which a non- or poorlyimmunogenic substance (e.g. a hapten) is naturally or artificiallylinked to enhance its immunogenicity.

Immortalization of antibody-producing cells can be carried out usingmethods which are well-known in the art. For example, theimmortalization may be achieved by the transformation method usingEpstein-Barr virus (EBV) (Kozbor et al., Methods in Enzymology 121: 140,1986). In another embodiment, antibody-producing cells are immortalizedusing the cell fusion method (described in Coligan et al., 1991-1997,supra), which is widely employed for the production of monoclonalantibodies. In this method, somatic antibody-producing cells with thepotential to produce antibodies, particularly B cells, are fused with amyeloma cell line. These somatic cells may be derived from the lymphnodes, spleens and peripheral blood of primed animals, preferably rodentanimals such as mice and rats. In a particular embodiment, mice spleencells can be used. In other embodiments, rat, rabbit, sheep or goatcells can also be used. Specialized myeloma cell lines have beendeveloped from lymphocytic tumours for use in hybridoma-producing fusionprocedures (Kohler and Milstein, 1976, supra; Shulman et al., Nature276: 269-270, 1978; Volk et al., J. Virol. 42(1): 220-227, 1982). Manymyeloma cell lines can also be used for the production of fused cellhybrids, including, e.g. P3.times.63-Ag8, P3.times.63-AG8.653,P3/NS1-Ag-4-1 (NS-1), Sp2/0-Ag14 and S194/5.XXO.Bu.1. TheP3.times.63-Ag8 and NS-1 cell lines have been described by Kohler andMilstein (1976, supra). Shulman et al. (1978, supra) developed theSp210-Ag14 myeloma line. The S194/5.XXO.Bu.1 line was reported byTrowbridge (J. Exp. Med. 148(1): 313-323, 1978). Methods for generatinghybrids of antibody-producing spleen or lymph node cells and myelomacells usually involve mixing somatic cells with myeloma cells in a 10:1proportion (although the proportion may vary from about 20:1 to about1:1), respectively, in the presence of an agent or agents (chemical,viral or electrical) that promotes the fusion of cell membranes. Fusionmethods have been described (Kohler and Milstein, 1975, supra; Kohlerand Milstein, 1976, supra; Gefter et al., Somatic Cell Genet. 3:231-236, 1977; Volk et al., 1982, supra). The fusion-promoting agentsused by those investigators were Sendai virus and polyethylene glycol(PEG). In certain embodiments, means to select the fused cell hybridsfrom the remaining unfused cells, particularly the unfused myelomacells, are provided. Generally, the selection of fused cell hybrids canbe accomplished by culturing the cells in media that support the growthof hybridomas but prevent the growth of the unfused myeloma cells, whichnormally would go on dividing indefinitely. The somatic cells used inthe fusion do not maintain long-term viability in in vitro culture andhence do not pose a problem. Several weeks are required to selectivelyculture the fused cell hybrids. Early in this time period, it isnecessary to identify those hybrids which produce the desired antibody,so that they may subsequently be cloned and propagated. Generally,around 10% of the hybrids obtained produce the desired antibody,although a range of from about 1 to about 30% is not uncommon. Thedetection of antibody-producing hybrids can be achieved by any one ofseveral standard assay methods, including enzyme-linked immunoassay andradioimmunoassay techniques as, for example, described in Kennet et al.(Monoclonal Antibodies and Hybridomas: A New Dimension in BiologicalAnalyses, pp 376-384, Plenum Press, New York, 1980) and by FACS analysis(O'Reilly et al., Biotechniques 25: 824-830, 1998).

Once the desired fused cell hybrids have been selected and cloned intoindividual antibody-producing cell lines, each cell line may bepropagated in either of two standard ways. A suspension of the hybridomacells can be injected into a histocompatible animal. The injected animalwill then develop tumours that secrete the specific monoclonal antibodyproduced by the fused cell hybrid. The body fluids of the animal, suchas serum or ascites fluid, can be tapped to provide monoclonalantibodies in high concentration. Alternatively, the individual celllines may be propagated in vitro in laboratory culture vessels. Theculture medium containing high concentrations of a single specificmonoclonal antibody can be harvested by decantation, filtration orcentrifugation, and subsequently purified.

The cell lines can then be tested for their specificity to detect theAkt kinase of interest by any suitable immunodetection means. Forexample, cell lines can be aliquoted into a number of wells andincubated and the supernatant from each well is analyzed byenzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibodytechnique, or the like. The cell line(s) producing a monoclonal antibodycapable of recognizing the target LIM kinase but which does notrecognize non-target epitopes are identified and then directly culturedin vitro or injected into a histocompatible animal to form tumours andto produce, collect and purify the required antibodies.

The invention encompasses, therefore, a method of detecting in a samplean Akt kinase or fragment, variant or derivative thereof includingcontacting the sample with an antibody or fragment or derivative thereofand detecting the level of a complex containing the antibody and Aktkinase or fragment, variant or derivative thereof compared to normalcontrols wherein elevated levels of Akt kinase is determined. Anysuitable technique for determining formation of the complex may be used.For example, an antibody according to the invention, having a reportermolecule associated therewith, may be utilized in immunoassays. Suchimmunoassays include but are not limited to radioimmunoassays (RIAs),enzyme-linked immunosorbent assays (ELISAs) immunochromatographictechniques (ICTs), and Western blotting which are well known to those ofskill in the art. Immunoassays can also include competitive assays. Thepresent invention encompasses qualitative and quantitative immunoassays.

Suitable immunoassay techniques are described, for example, in U.S. Pat.Nos. 4,016,043; 4,424,279; and 4,018,653. These include both single-siteand two-site assays of the non-competitive types, as well as thetraditional competitive binding assays. These assays also include directbinding of a labeled antigen-binding molecule to a target antigen.

The invention further provides methods for quantifying Akt proteinexpression and activation levels in cells or tissue samples obtainedfrom an animal, such as a human cancer patient or an individualsuspected of having cancer. In one embodiment, the invention providesmethods for quantifying Akt protein expression or activation levelsusing an imaging system quantitatively. The imaging system can be usedto receive, enhance, and process images of cells or tissue samples, thathave been stained with AKT protein-specific stains, in order todetermine the amount or activation level of AKT protein expressed in thecells or tissue samples from such an animal. In embodiments of themethods of the invention, a calibration curve of AKT1 and AKT2 proteinexpression can be generated for at least two cell lines expressingdiffering amounts of AKT protein. The calibration curve can then used toquantitatively determine the amount of AKT protein that is expressed ina cell or tissue sample. Analogous calibration curves can be made foractivated AKT proteins using reagents specific for the activationfeatures. It can also be used to determine changes in amounts andactivation state of AKT before and after clinical cancer treatment.

In one particular embodiment of the methods of the invention, AKTprotein expression in a cell or tissue sample can be quantified using anenzyme-linked immunoabsorbent assay (ELISA) to determine the amount ofAKT protein in a sample. Such methods are described, for example, inU.S. Patent Publication No. 2002/0015974.

In other embodiments enzyme immunoassays can be used to detect the Aktkinase. In such assays, an enzyme is conjugated to the second antibody,generally by means of glutaraldehyde or periodate. The substrates to beused with the specific enzymes are generally chosen for the productionof, upon hydrolysis by the corresponding enzyme, a detectable colourchange. It is also possible to employ fluorogenic substrates, whichyield a fluorescent product rather than the chromogenic substrates. Theenzyme-labeled antibody can be added to the first antibody-antigencomplex, allowed to bind, and then the excess reagent washed away. Asolution containing the appropriate substrate can then be added to thecomplex of antibody-antigen-antibody. The substrate can react with theenzyme linked to the second antibody, giving a qualitative visualsignal, which may be further quantitated, usuallyspectrophotometrically, to give an indication of the amount of antigenwhich was present in the sample. Alternately, fluorescent compounds,such as fluorescein, rhodamine and the lanthanide, europium (EU), can bechemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labeled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic colour visually detectable with a lightmicroscope. The fluorescent-labeled antibody is allowed to bind to thefirst antibody-antigen complex. After washing off the unbound reagent,the remaining tertiary complex is then exposed to light of anappropriate wavelength. The fluorescence observed indicates the presenceof the antigen of interest. Immunofluorometric assays (IFMA) are wellestablished in the art and are particularly useful for the presentmethod. However, other reporter molecules, such as radioisotope,chemiluminescent or bioluminescent molecules can also be employed.

In a particular embodiment, antibodies to Akt kinase can also be used inELISA-mediated detection of Akt kinase especially in serum or othercirculatory fluid. This can be accomplished by immobilizing anti-Aktkinase antibodies to a solid support and contacting these with abiological extract such as serum, blood, lymph or other bodily fluid,cell extract or cell biopsy. Labeled anti-Akt kinase antibodies can thenbe used to detect immobilized Akt kinase. This assay can be varied inany number of ways and all variations are encompassed by the presentinvention and known to one skilled in the art. This approach can enablerapid detection and quantitation of Akt kinase levels using, forexample, a serum-based assay.

In one embodiment, an Akt Elisa assay kit may be used in the presentinvention. For example, a Cellular Activation of Signaling ELISA kit forAkt S473 from SuperArray Bioscience can be utilized in the presentinvention. In one embodiment, the antibody can be an anti-pan antibodythat recognizes Akt S473. Elisa assay kit containing an anti-Akiantibody and additional reagents, including, but not limited to, washingbuffer, antibody dilution buffer, blocking buffer, cell stainingsolution, developing solution, stop solution, secondary antibodies, anddistilled water.

Nucleotide Detection

In another embodiment, a method to detect Akt kinases is provided bydetecting the level of expression in a cell of a polynucleotide encodingan Akt kinase. Expression of the polynucleotide can be determined usingany suitable technique known to one skilled in the art. In oneembodiment, a labeled polynucleotide encoding an Akt kinase can beutilized as a probe in a Northern blot of an RNA extract obtained fromthe cell. In other embodiments, a nucleic acid extract from an animalcan be utilized in concert with oligonucleotide primers corresponding tosense and antisense sequences of a polynucleotide encoding the kinase,or flanking sequences thereof, in a nucleic acid amplification reactionsuch as RT PCR. A variety of automated solid-phase detection techniquesare also available to one skilled in the art, for example, as describedby Fodor et al. (Science 251: 767-777, 1991) and Kazal et al. (NatureMedicine 2: 753-759, 1996).

In other embodiments, methods are provided to detect akt kinase encodingRNA transcripts. The RNA can be isolated from a cellular samplesuspected of containing Akt kinase RNA, e.g. total RNA isolated fromhuman cancer tissue. RNA can be isolated by methods known in the art,e.g. using TRIZOL reagent (GIBCO-BRL/Life Technologies, Gaithersburg,Md.). Oligo-dT, or random-sequence oligonucleotides, as well assequence-specific oligonucleotides can be employed as a primer in areverse transcriptase reaction to prepare first-strand cDNAs from theisolated RNA. Resultant first-strand cDNAs can then amplified withsequence-specific oligonucleotides in PCR reactions to yield anamplified product.

Polymerase chain reaction or “PCR” refers to a procedure or technique inwhich amounts of a preselected fragment of nucleic acid, RNA and/or DNA,are amplified as described, for example, in U.S. Pat. No. 4,683,195.Generally, sequence information from the ends of the region of interestor beyond is employed to design oligonucleotide primers. These primerswill be identical or similar in sequence to opposite strands of thetemplate to be amplified. PCR can be used to amplify specific RNAsequences and cDNA transcribed from total cellular RNA. See generallyMullis et al. (Quant. Biol. 51: 263, 1987; Erlich, eds., PCR Technology,Stockton Press, NY, 1989). Thus, amplification of specific nucleic acidsequences by PCR relies upon oligonucleotides or “primers” havingconserved nucleotide sequences wherein the conserved sequences arededuced from alignments of related gene or protein sequences, e.g. asequence comparison of mammalian Akt kinase genes. For example, oneprimer is prepared which is predicted to anneal to the antisense strandand another primer prepared which is predicted to anneal to the sensestrand of a cDNA molecule which encodes a Akt kinase. To detect theamplified product, the reaction mixture is typically subjected toagarose gel electrophoresis or other convenient separation technique andthe relative presence of the Akt kinase specific amplified DNA detected.For example, Akt kinase amplified DNA may be detected using Southernhybridization with a specific oligonucleotide probe or comparing itselectrophoretic mobility with DNA standards of known molecular weight.Isolation, purification and characterization of the amplified Akt kinaseDNA can be accomplished by excising or eluting the fragment from the gel(for example, see references Lawn et al., Nucleic Acids Res. 2: 6103,1981; Goeddel et al., Nucleic acids Res. 8: 4057-1980), cloning theamplified product into a cloning site of a suitable vector, such as thepCRII vector (Invitrogen), sequencing the cloned insert and comparingthe DNA sequence to the known sequence of LIM kinase. The relativeamounts of LIMN kinase mRNA and cDNA can then be determined.

In one embodiment, real-time PCR can be used to determinetranscriptional levels of Akt nucleotides. Determination oftranscriptional activity also includes a measure of potentialtranslational activity based on available mRNA transcripts. Real-timePCR as well as other PCR procedures use a number of chemistries fordetection of PCR product including the binding of DNA bindingfluorophores, the 5′ endonuclease, adjacent liner and hairpinoligoprobes and the self-fluorescing amplicons. These chemistries andreal-time PCR in general are discussed, for example, in Mackay et al.Nucleic Acids Res 30(6): 1292-1305, 2002; Walker, J. Biochem. Mol.Toxicology. 15(3): 121-127, 2001; Lewis et al., J. Pathol. 195: 66-71,2001.

In an alternate embodiment, the aberrant expression of Akt can beidentified by contacting a nucleotide sequences isolated from abiological sample with an oligonucleotide probe having a sequencecomplementary to an Akt sequences selected from the nucleotide sequencesof FIGS. 6 a-c, 7 a-d, or 8 a-c, or fragment thereof, and then detectingthe sequence by hybridizing the probe to the sequence, and comparing theresults to a normal sample. The hybridization of the probe to thebiological sample can be detected by labeling the probe using anydetectable agent. The probe can be labeled for example, with aradioisotope, or with biotin, fluorescent dye, electron-dense reagent,enzyme, hapten or protein for which antibodies are available. Thedetectable label can be assayed by any desired means, includingspectroscopic, photochemical, biochemical, immunochemical,radioisotopic, or chemical means. The probe can also be detected usingtechniques such as an oligomer restriction technique, a dot blot assay,a reverse dot blot assay, a line probe assay, and a 5′ nuclease assay.Alternatively, the probe can be detected using any of the generallyapplicable DNA array technologies, including macroarray, microarray andDNA microchip technologies. The oligonucleotide probe typically includesapproximately at least 14, 15, 16, 18, 20, 25 or 28 nucleotides thathybridize to the nucleotides selected from FIGS. 6 a-c, 7 a-d, and 8a-c, or a fragment thereof. It is generally not preferred to use a probethat is greater than approximately 25 or 28 nucleotides in length. Theoligonucleotide probe is designed to identify an Akt nucleotidesequence.

Kinase Assays

The activity of the Akt kinases can be measured using any suitablekinase assay known in the art. For example, and not by way oflimitation, the methods described in Hogg et al (Oncogene 1994 9:98-96),Mills et al (J. Biol. Chem. 1992 267:16000-006) and Tomizawa et al 2001(FEBS Lett. 2001 492: 221-7), Schmandt et al, (J. Immunol. 1994.152:96-105) can be used. Further serine, threonine and tyrosine kinaseassays are described in Ausubel et al. (Short Protocols in MolecularBiology, 1999, unit 17.6).

Akt kinase assays can generally use an Akt polypeptide, a labeled donorsubstrate, and a receptor substrate that is either specific ornon-specific for Akt. In such assays Akt transfers a labeled moiety fromthe donor substrate to the receptor substrate, and kinase activity ismeasured by the amount of labeled moiety transferred from the donorsubstrate to the receptor substrate. Akt polypeptide can be producedusing various expression systems, can be purified from cells, can be inthe form of a cleaved or uncleaved recombinant fusion protein and/or canhave non-Akt polypeptide sequences, for example a His tag or.beta.-galactosidase at its N- or C-terminus. Akt activity can beassayed in cancerous cells lines if the cancerous cell lines are used asa source of the Akt to be assayed. Suitable donor substrates for Aktassays include any Fmolecule that is susceptible to dephosphorylation byAkt., such as, for example include .gamma.-labeled ATP and ATP analogs,wherein the label is ³³P, ³²P, ³⁵S or any other radioactive isotope or asuitable fluorescent marker. Suitable recipient substrates for Aktassays include any polypeptide or other molecule that is susceptible tophosphorylation by Akt. Recipient substrates can be derived fromfragments of in vivo targets of Akt. Recipient substrates fragments canbe 8 to 50 amino acids in length, usually 10 to 30 amino acids andparticularly of about 10, 12, 15, 18, 20 and 25 amino acids in length.Further recipient substrates can be determined empirically using a setof different polypeptides or other molecules. Targets of Recipientsubstrates for TTK can be capable of being purified from othercomponents of the reaction once the reaction has been performed. Thispurification is usually done through a molecular interaction, where therecipient substrates is biotinylated and purified through itsinteraction with streptavidin, or a specific antibody is available thatcan specifically recognize the recipient substrates. The reaction can beperformed in a variety of conditions, such as on a solid support, in agel, in solution or in living cells. The choice of detection methodsdepends on type of label used for the donor molecule and may include,for example, measurement of incorporated radiation or fluorescence byautoradiography, scintillation, scanning or fluorography.

6. METHODS OF TREATMENT

The compounds and pharmaceutical compositions provided herein can beused in the treatment of a condition including tumors, cancer, and otherdisorders associated with abnormal cell proliferation. In oneembodiment, the compounds of the present invention can be used to treata carcinoma, sarcoma, lymphoma, leukemia, and/or myeloma. In otherembodiments of the present invention, the compounds disclosed herein canbe used to treat solid tumors.

The compounds of the present invention invention can be used for thetreatment of cancer, such as, but not limited to cancer of the followingorgans or tissues: breast, prostate, lung, bronchus, colon, urinary,bladder, non-Hodgkin lymphoma, melanoma, kidney, renal, pancreas,pharnx, thyroid, stomach, brain, multiple myeloma, esophagus, liver,intrahepatic bile duct, cervix, larynx, acute myeloid leukemia, chroniclymphatic leukemia, soft tissue, such as heart, Hodgkin lymphoma,testis, small intestine, chronic myeloid leukemia, acute lymphaticleukemia, anus, anal canal, anorectal, thyroid, vulva, gallbladder,pleura, eye, nose nasal cavity, middle ear, nasopharnx, ureter,peritoneum, omentum, mesentery, and gastrointestineal, high gradeglioma, glioblastoma, colon, rectal, pancreatic, gastric cancers,hepatocellular carcinoma; head and neck cancers, carcinomas; renal cellcarcinoma; adenocarcinoma; sarcomas; hemangioendothelioma; lymphomas;leukemias, mycosis iungoides. In additional embodiments, the compoundsof the invention can be used to treat skin diseases including, but notlimited to, the malignant diseases angiosarcoma, hemangioendothelioma,basal cell carcinoma, squamous cell carcinoma, malignant melanoma andKaposi's sarcoma, and the non-malignant diseases or conditions such aspsoriasis, lymphangiogenesis, hemangioma of childhood, Sturge-Webersyndrome, verruca vulgaris, neurofibromatosis, tuberous sclerosis,pyogenic granulomas, recessive dystrophic epidermolysis bullosa, venousulcers, acne, rosacea, eczema, molluscum contagious, seborrheickeratosis, and actinic keratosis.

Compositions including the compounds of the invention can be used totreat these cancers and other cancers at any stage from the discovery ofthe cancer to advanced stages. In addition, compositions includingcompounds of the invention can be used in the treatment of the primarycancer and metastases thereof.

In other embodiments of the invention, the compounds described hereincan be used for the treatment of cancer, including, but not limited to,the cancers listed in Table 1 below.

TABLE 1 Types of Cancer Acute Lymphoblastic Leukemia, Adult AcuteLymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, Adult AcuteMyeloid Leukemia, Childhood Adrenocortical Carcinoma AdrenocorticalCarcinoma, Childhood AIDS-Related Cancers AIDS-Related Lymphoma AnalCancer Astrocytoma, Childhood Cerebellar Astrocytoma, Childhood CerebralBasal Cell Carcinoma Bile Duct Cancer, Extrahepatic Bladder CancerBladder Cancer, Childhood Bone Cancer, Osteosarcoma/Malignant FibrousHistiocytoma Brain Stem Glioma, Childhood Brain Tumor, Adult BrainTumor, Brain Stem Glioma, Childhood Brain Tumor, Cerebellar Astrocytoma,Childhood Brain Tumor, Cerebral Astrocytoma/Malignant Glioma, ChildhoodBrain Tumor, Ependymoma, Childhood Brain Tumor, Medulloblastoma,Childhood Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors,Childhood Brain Tumor, Visual Pathway and Hypothalamic Glioma, ChildhoodBrain Tumor, Childhood Breast Cancer Breast Cancer, Childhood BreastCancer, Male Bronchial Adenomas/Carcinoids, Childhood Burkitt's LymphomaCarcinoid Tumor, Childhood Carcinoid Tumor, Gastrointestinal Carcinomaof Unknown Primary Central Nervous System Lymphoma, Primary CerebellarAstroeytoma, Childhood Cerebral Astrocytoma/Malignant Glioma, ChildhoodCervical Cancer Childhood Cancers Chronic Lymphocytic Leukemia ChronicMyelogenous Leukemia Chronic Myeloproliferative Disorders Colon CancerColorectal Cancer, Childhood Cutaneous T-Cell Lymphoma, see MycosisFungoides and Sézary Syndrome Endometrial Cancer Ependymoma, ChildhoodEsophageal Cancer Esophageal Cancer, Childhood Ewing's Family of TumorsExtracranial Germ Cell Tumor, Childhood Extragonadal Germ Cell TumorExtrahepatic Bile Duct Cancer Eye Cancer, Intraocular Melanoma EyeCancer, Retinoblastoma Gallbladder Cancer Gastric (Stomach) CancerGastric (Stomach) Cancer, Childhood Gastrointestinal Carcinoid TumorGerm Cell Tumor, Extracranial, Childhood Germ Cell Tumor, ExtragonadalGerm Cell Tumor, Ovarian Gestational Trophoblastic Tumor Glioma, AdultGlioma, Childhood Brain Stem Glioma, Childhood Cerebral AstrocytomaGlioma, Childhood Visual Pathway and Hypothalamic Skin Cancer (Melanoma)Skin Carcinoma, Merkel Cell Small Cell Lung Cancer Small IntestineCancer Soft Tissue Sarcoma, Adult Soft Tissue Sarcoma, ChildhoodSquamous Cell Carcinoma, See Skin Cancer (non-Melanoma) Squamous NeckCancer with Occult Primary, Metastic Stomach (Gastric) Cancer Stomach(Gastric) Cancer, Childhood Supratentorial Primitive NeurocctodermalTumors, Childhood T-Cell Lymphoma, Cutaneous, see Mycosis Fungoides andSézary Syndrome Testicular Cancer Thymoma, Childhood Thymoma and ThymicCarcinoma Thyroid Cancer Thyroid Cancer, Childhood Transitional CellCancer of the Renal Pelvis and Ureter Trophoblastic Tumor, GestationalUnknown Primary Site, Carcinoma of, Adult Unknown Primary Site, Cancerof, Childhood Unusual Cancers of Childhood Ureter and Renal Pelvis,Transitional Cell Cancer Urethral Cancer Uterine Cancer, EndometrialUterine Sarcoma Vaginal Cancer Visual Pathway and Hypothalamic Glioma,Childhood Vulvar Cancer Waldentröm's Macroglobulinemia Wilms' TumorHairy Cell Leukemia Head and Neck Cancer Hepatocellular (Liver) Cancer,Adult (Primary) Hepatocellular (Liver) Cancer, Childhood (Primary)Hodgkin's Lymphoma, Adult Hodgkin's Lymphoma, Childhood Hodgkin'sLymphoma During Pregnancy Hypopharyngeal Cancer Hypothalamic and VisualPathway Glioma, Childhood Intraocular Melanoma Islet Cell Carcinoma(Endocrine Pancreas) Kaposi's Sarcoma Kidney (Renal Cell) Cancer KidneyCancer, Childhood Laryngeal Cancer Laryngeal Cancer, Childhood Leukemia,Acute Lymphoblastic, Adult Leukemia, Acute Lymphoblastic, ChildhoodLeukemia, Acute Myeloid, Adult Leukemia, Acute Myeloid, ChildhoodLeukemia, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, BCell Lip and Oral Cavity Cancer Liver Cancer, Adult (Primary) LiverCancer, Childhood (Primary) Lung Cancer, Non-Small Cell Lung Cancer,Small Cell Lymphoma, AIDS-Related Lymphoma, Burkitt's Lymphoma,Cutaneous T-Cell, see Mycosis Fungoides and Sézary Syndrome Lymphoma,Hodgkin's, Adult Lymphoma, Hodgkin's, Childhood Lymphoma, Hodgkin'sDuring Pregnancy Lymphoma, Non-Hodgkin's, Adult Lymphoma, Non-Hodgkin's,Childhood Lymphoma, Non-Hodgkin's During Pregnancy Lymphoma, PrimaryCentral Nervous System Macroglobulinemia, Waldenström's MalignantFibrous Histiocytoma of Bone/Osteosarcoma Medulloblastoma, ChildhoodMelanoma Melanoma, Intraocular (Eye) Merkel Cell Carcinoma Mesothelioma,Adult Malignant Mesothelioma, Childhood Metastatic Squamous Neck Cancerwith Occult Primary Multiple Endocrine Neoplasia Syndrome, ChildhoodMultiple Myeloma/Plasma Cell Neoplasm Mycosis Fungoides MyelodysplasticSyndromes Myelodysplastic/Myeloproliferative Diseases MyelogenousLeukemia, Chronic Myeloid Leukemia, Adult Acute Myeloid Leukemia,Childhood Acute Myeloma, Multiple Myeloproliferative Disorders, ChronicNasal Cavity and Paranasal Sinus Cancer Nasopharyngeal CancerNasopharyngeal Cancer, Childhood Neuroblastoma Non-Hodgkin's Lymphoma,Adult Non-Hodgkin's Lymphoma, Childhood Non-Hodgkin's Lymphoma DuringPregnancy Non-Small Cell Lung Cancer Oral Cancer, Childhood Oral CavityCancer, Lip and Oropharyngeal Cancer Osteosarcoma/Malignant FibrousHistiocytoma of Bone Ovarian Cancer, Childhood Ovarian Epithelial CancerOvarian Germ Cell Tumor Ovarian Low Malignant Potential Tumor PancreaticCancer Pancreatic Cancer, Childhood Pancreatic Cancer, Islet CellParanasal Sinus and Nasal Cavity Cancer Parathyroid Cancer Penile CancerPheochromocytoma Pineoblastoma and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Pituitary Tumor Plasma CellNeoplasm/Multiple Myeloma Pleuropulmonary Blastoma Pregnancy and BreastCancer Pregnancy and Hodgkin's Lymphoma Pregnancy and Non-Hodgkin'sLymphoma Primary Central Nervous System Lymphoma Prostate Cancer RectalCancer Renal Cell (Kidney) Cancer Renal Cell (Kidney) Cancer, ChildhoodRenal Pelvis and Ureter, Transitional Cell Cancer RetinoblastomaRhabdomyosarcoma, Childhood Salivary Gland Cancer Salivary Gland Cancer,Childhood Sarcoma, Ewing's Family of Tumors Sarcoma, Kaposi's Sarcoma,Soft Tissue, Adult Sarcoma, Soft Tissue, Childhood Sarcoma, UterineSezary Syndrome Skin Cancer (non-Melanoma) Skin Cancer, Childhood

In further embodiments of the present invention, the compounds disclosedherein can be used in the treatment of angiogenesis-related diseases.

Antiangiogenic small molecules include thalidomide, which acts in partby inhibiting NFkB, 2-methoxyestradiol, which influences microtubuleactivation and hypoxia inducing factor (HIF1a) activation,cyclo-oxygenase 2 (COX2) inhibitors, and low doses of conventionalchemotherapeutic agents, including cyclophosphamide, taxanes,anthracycline analogs, and vinca alkaloids (vincristine, vinblastine)(D'Amato, R. J. et al., (1994) Proc. Natl. Acad. Sci. U.S. A 91,3964-3968, D'Amato, R. J. et al. (1994) Proc. Natl. Acad. Sci. U.S. A91, 4082-4085). In addition, certain tyrosine kinase inhibitorsindirectly decrease angiogenesis by decreasing production of VEGF andother proangiogenic factors by tumor and stromal cells. These drugsinclude Herceptin, imatinib (Glivec), and Iressa (Bergers, G. et al.(2003) Journal of Clinical Investigation 111, 1287-1295, Ciardiello, F.et al. (2001) Clinical Cancer Research 7, 1459-1465, Plum, S. M. et al.(2003) Clinical Cancer Research 9, 4619-4626).

Recently, angiogenesis inhibitors have moved from animal models to humanpatients. Angiogenesis inhibitors represent a promising treatment for avariety of cancers. Recently, Avastin a high affinity antibody againstvascular endothelial growth factor (VEGF), has been shown to prolonglife as a single agent in advanced renal cell carcinoma and prolong lifein combination with chemotherapy in advanced colon cancer (Yang, J. C.et al. (2003) New England Journal of Medicine 349, 427-434, Kabbinavar,F. et al. (2003) Journal of Clinical Oncology 21, 60-65).

Angiogenesis-related diseases include, but are not limited to,inflammatory, autoimmune, and infectious diseases;angiogenesis-dependent cancer, including, for example, solid tumors,blood born tumors such as leukemias, and tumor metastases; benigntumors, for example hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis;eczema; ocular angiogenic diseases, for example, diabetic retinopathy,retinopathy of prematurity, macular degeneration, corneal graftrejection, neovascular glaucoma, retrolental libroplasia, rubeosis;Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;and wound granulation. In addition, compositions of this invention canbe used to treat diseases such as, but not limited to, intestinaladhesions, atherosclerosis, scleroderma, warts, and hypertrophic scars(i.e., keloids). Compositions of this invention can also be used in thetreatment of diseases that have angiogenesis as a pathologic consequencesuch as cat scratch disease (Rochele minalia quintosa), ulcers(Helobacter pylori), tuberculosis, and leprosy.

6.1. Treatment of Drug Resistant Tumors or Cancers

The invention provides compounds that can be used to treat drugresistant cancer, including the embodiments of cancers and the TCN,TCN-P, TCN-PM and/or related compounds and/or one or more additionalanti-cancer agents disclosed herein.

Multidrug resistance (MDR) occurs in human cancers and can be asignificant obstacle to the success of chemotherapy. Multidrugresistance is a phenomenon whereby tumor cells in vitro that have beenexposed to one cytotoxic agent develop cross-resistance to a range ofstructurally and functionally unrelated compounds. In addition, MDR canoccur intrinsically in some cancers without previous exposure tochemotherapy agents. Thus, in one embodiment, the present inventionprovides methods for the treatment of a patient with a drug resistantcancer, for example, multidrug resistant cancer, by administration ofTCN, TCN-P, TCN-PM and/or related compounds and one or more additionalanti-cancer agents as disclosed herein. In certain embodiments, TCN,TCN-P, TCN-PM and/or related compounds and one or more additionalanti-cancer agents can be used to treat cancers that are resistant totaxol alone, rapamycin, tamoxifen, cisplatin, and/or gefitinib (iressa).

In one embodiment, TCN, TCN-P, TCN-PM and/or related compounds and oneor more additional anti-cancer agents as disclosed herein can be usedfor the treatment of drug resistant cancers of the colon, bone, kidney,adrenal, pancreas, liver and/or any other cancer known in the art ordescribed herein.

The invention encompasses compounds that can be used to treat drugresistant cancer, including the embodiments of cancers disclosed hereinby administering TCN, TCN-P, TCN-PM and/or related compounds and one ormore taxanes.

In one embodiment, the invention encompasses methods for the treatmentof a patient with a drug resistant cancer, for example, multidrugresistant cancer by administration of TCN, TCN-P, TCN-PM and/or relatedcompounds and one or more taxanes. In certain embodiments, TCN, TCN-P,TCN-PM and/or related compounds and one or more taxanes can be used totreat cancers that are resistant to taxol alone, rapamycin, tamoxifen,cisplatin, and/or gefitinib (iressa).

In one embodiment, TCN, TCN-P, TCN-PM and/or related compounds and oneor more taxanes can be used for the treatment of drug resistant cancersof the colon, bone, kidney, adrenal, pancreas, liver and/or any othercancer known in the art or described herein.

The invention encompasses compounds that can be used to treat drugresistant cancer, including the embodiments of cancers disclosed hereinby administering TCN, TCN-P, TCN-PM and/or related compounds and amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof.

In one embodiment, the invention encompasses methods for the treatmentof a patient with a drug resistant cancer, for example, multidrugresistant cancer by administration of TCN, TCN-P, TCN-PM and/or relatedcompounds and a molecule that modulates the HER2/neu (erbB2) receptor,for example, trastuzumab or a salt thereof. In certain embodiments, TCN,TCN-P, TCN-PM and/or related compounds and a molecule that modulates theHER2/neu (erbB2) receptor, for example, trastuzumab or a salt thereof,can be used to treat cancers that are resistant to taxol alone,rapamycin, tamoxifen, cisplatin, and/or gefitinib (iressa).

In one embodiment, TCN, TCN-P, TCN-PM and/or related compounds and amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, can be used for the treatment of drugresistant cancers of the colon, bone, kidney, adrenal, pancreas, liverand/or any other cancer known in the art or described herein.

The invention encompasses compounds that can be used to treat drugresistant cancer, including the embodiments of cancers disclosed hereinby administering TCN, TCN-P, TCN-PM and/or related compounds and amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof.

In one embodiment, the invention encompasses methods for the treatmentof a patient with a drug resistant cancer, for example, multidrugresistant cancer by administration of TCN, TCN-P, TCN-PM and/or relatedcompounds and one or more anthracycline analogs. In certain embodiments,TCN, TCN-P, TCN-PM and/or related compounds and one or moreanthracycline analogs can be used to treat cancers that are resistant totaxol alone, rapamycin, tamoxifen, cisplatin, and/or gefitinib (iressa).

In one embodiment, TCN, TCN-P, TCN-PM and/or related compounds and oneor more anthracycline analogs can be used for the treatment of drugresistant cancers of the colon, bone, kidney, adrenal, pancreas, liverand/or any other cancer known in the art or described herein.

The invention encompasses compounds that can be used to treat drugresistant cancer, including the embodiments of cancers disclosed hereinby administering TCN, TCN-P, TCN-PM and/or related compounds and anerlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof.

In one embodiment, the invention encompasses methods for the treatmentof a patient with a drug resistant cancer, for example, multidrugresistant cancer by administration of TCN, TCN-P, TCN-PM and/or relatedcompounds and an erlotinib-like compound, for example, gefitinib,erlotinib or a salt thereof. In certain embodiments, TCN, TCN-P, TCN-PMand/or related compounds and an erlotinib-like compound, for example,gefitinib, erlotinib or a salt thereof can be used to treat cancers thatare resistant to taxol alone, rapamycin, tamoxifen, cisplatin, and, orgefitinib (iressa).

In one embodiment, TCN, TCN-P, TCN-PM and/or related compounds and anerlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof can be used for the treatment of drug resistant cancers of thecolon, bone, kidney, adrenal, pancreas, liver and/or any other cancerknown in the art or described herein.

The invention encompasses compounds that can be used to treat drugresistant cancer, including the embodiments of cancers disclosed hereinby administering TCN, TCN-P, TCN-PM and/or related compounds and one ormore platinum compounds.

In one embodiment, the invention encompasses methods for the treatmentof a patient with a drug resistant cancer, for example, multidrugresistant cancer by administration of TCN, TCN-P, TCN-PM and/or relatedcompounds and one or more platinum compounds. In certain embodiments,TCN, TCN-P, TCN-PM and/or related compounds and one or more platinumcompounds can be used to treat cancers that are resistant to taxolalone, rapamycin, tamoxifen, cisplatin, and/or gefitinib (iressa).

In one embodiment, TCN, TCN-P, TCN-PM and/or related compounds and oneor more platinum compounds can be used for the treatment of drugresistant cancers of the colon, bone, kidney, adrenal, pancreas, liverand/or any other cancer known in the art or described herein.

The invention encompasses compounds that can be used to treat drugresistant cancer, including the embodiments of cancers disclosed hereinby administering TCN, TCN-P, TCN-PM and/or related compounds andbortezomib or a salt thereof.

In one embodiment, the invention encompasses methods for the treatmentof a patient with a drug resistant cancer, for example, multidrugresistant cancer by administration of TCN. TCN-P, TCN-PM and/or relatedcompounds and bortezomib or a salt thereof. In certain embodiments, TCN,TCN-P, TCN-PM and/or related compounds and bortezomib or a salt thereofcan be used to treat cancers that are resistant to taxol alone,rapamycin. tamoxifen, cisplatin, and or gefitinib (iressa).

In one embodiment, TCN, TCN-P, TCN-PM and/or related compounds andbortezomib or a salt thereof can be used for the treatment of drugresistant cancers of the colon, bone, kidney, adrenal, pancreas, liverand/or any other cancer known in the art or described herein.

6.2. Combination Therapy

In certain embodiments, the TCN, TCN-P, TCN-PM and/or related compoundsand one or more first additional anti-cancer agents of the invention canbe administered together with other cytotoxic agents. In anotherembodiment, the TCN, TCN-P, TCN-PM and/or related compounds and one ormore first additional anti-cancer agents and compositions thereof, whenused in the treatment of solid tumors, can be administered inconjunction with the use of radiation.

In another embodiment of the present invention, the TCN, TCN-P, TCN-PMand/or related compounds and one or more additional anti-cancer agentsand compositions disclosed herein can be combined with at least onesecond additional chemotherapeutic agent. The second additional agentscan be administered in combination or alternation with the compoundsdisclosed herein. The drugs can form part of the same composition, or beprovided as a separate composition for administration at the same timeor a different time.

In one embodiment, the TCN, TCN-P, TCN-PM and/or related compounds andone or more additional anti-cancer agents disclosed herein can becombined with antiangiogenic agents to enhance their effectiveness, orcombined with other antiangiogenic agents and administered together withother cytotoxic agents. In another embodiment, the TCN, TCN-P, TCN-PMand/or related compounds and taxanes and compositions, when used in thetreatment of solid tumors, can be administered with the agents selectedfrom, but not limited to IL-12, retinoids, interferons, angiostatin,endostatin, thalidomide, thrombospondin-1, thrombospondin-2, captopryl,anti-neoplastic agents such as alpha interferon, COMP (cyclophosphamide,vincristine, methotrexate and prednisone), etoposide, mBACOD(methortrexate, bleomycin, doxorubicin, cyclophosphamide, vincristineand dexamethasone), PRO-MACE/MOPP (prednisone, methotrexate (w/leucovinrescue), doxorubicin, cyclophosphamide, etoposide/mechlorethamine,vincristine, prednisone and procarbazine), vincristine, vinblastine,angioinhibins, TNP-470, pentosan polysulfate, platelet factor 4,angiostatin, LM-609, SU-101, CM-101, Techgalan, thalidomide, SP-PG andradiation. In further embodiments, the compounds and compositionsdisclosed herein can be administered in combination or alternation with,for example, drugs with antimitotic effects, such as those which targetcytoskeletal elements, including podophyllotoxins or vinca alkaloids(vincristine, vinblastine); antimetabolite drugs (such as5-fluorouracil, cytarabine, gemcitabine, purine analogues such aspentostatin, methotrexate); alkylating agents or nitrogen mustards (suchas nitrosoureas, cyclophosphamide or ifosphamide); drugs which targetDNA such as the anthracycline drugs adriamycin, doxorubicin,pharmorubicin or epirubicin; drugs which target topoisomerases such asetoposide; hormones and hormone agonists or antagonists such asestrogens, antiestrogens (tamoxifen and related compounds) andandrogens, flutamide, leuprorelin, goserelin, cyproterone or octreotide;drugs which target signal transduction in tumour cells includingantibody derivatives such as herceptin; alkylating drugs such asplatinum drugs (cisplatin, carbonplatin, oxaliplatin, paraplatin) ornitrosoureas; drugs potentially affecting metastasis of tumours such asmatrix metalloproteinase inhibitors; gene therapy and antisense agents;antibody therapeutics; other bioactive compounds of marine origin,notably the didemnins such as aplidine; steroid analogues, in particulardexamethasone; anti-inflammatory drugs, including nonsteroidal agents(such as acetaminophen or ibuprofen) or steroids and their derivativesin particular dexamethasone; anti-emetic drugs, including 5HT-3inhibitors (such as gramisetron or ondasetron), and steroids and theirderivatives in particular dexamethasone. In still further embodiments,the compounds and compositions can be used in combination or alternationwith the chemotherapeutic agents disclosed below in Table 2.

TABLE 2 Chemotherapeutic Agents 13-cis-Retinoic Acid Neosar2-Amino-6-Mercaptopurine Neulasta 2-CdA Neumega 2-ChlorodeoxyadenosineNeupogen 5-fluorouracil Nilandron 5-FU Nilutamide 6-TG Nitrogen Mustard6-Thioguanine Novaldex 6-Mercaptopurine Novantrone 6-MP OctreotideAccutane Octreotide acetate Actinomycin-D Oncospar Adriamycin OncovinAdrucil Ontak Agrylin Onxal Ala-Cort Oprevelkin Aldesleukin OrapredAlemtuzumab Orasone Alitretinoin Oxaliplatin Alkaban-AQ PaclitaxelAlkeran Pamidronate All-transretinoic acid Panretin Alpha interferonParaplatin Altretamine Pediapred Amethopterin PEG Interferon AmifostinePegaspargase Aminoglutethimide Pegfilgrastim Anagrelide PEG-INTRONAnandron PEG-L-asparaginase Anastrozole Phenylalanine MustardArabinosylcytosine Platinol Ara-C Platinol-AQ Aranesp PrednisoloneAredia Prednisone Arimidex Prelone Aromasin Procarbazine Arsenictrioxide PROCRIT Asparaginase Proleukin ATRA Prolifeprospan 20 withCarmustine implant Avastin Purinethol BCG Raloxifene BCNU RheumatrexBevacizumab Rituxan Bexarotene Rituximab Bicalutamide Roveron-A(interferon alfa-2a) BiCNU Rubex Blenoxane Rubidomycin hydrochlorideBleomycin Sandostatin Bortezomib Sandostatin LAR Busulfan SargramostimBusulfex Solu-Cortef C225 Solu-Medrol Calcium Leucovorin STI-571 CampathStreptozocin Camptosar Tamoxifen Camptothecin-11 Targretin CapecitabineTaxol Carac Taxotere Carboplatin Temodar Carmustine TemozolomideCarmustine wafer Teniposide Casodex TESPA CCNU Thalidomide CDDP ThalomidCeeNU TheraCys Cerubidine Thioguanine cetuximab Thioguanine TabloidChlorambucil Thiophosphoamide Cisplatin Thioplex Citrovorum FactorThiotepa Cladribine TICE Cortisone Toposar Cosmegen Topotecan CPT-11Toremifene Cyclophosphamide Trastuzumab Cytadren Tretinoin CytarabineTrexall Cytarabine liposomal Trisenox Cytosar-U TSPA Cytoxan VCRDacarbazine Velban Dactinomycin Velcade Darbepoetin alfa VePesidDaunomycin Vesanoid Daunorubicin Viadur Daunorubicin hydrochlorideVinblastine Daunorubicin liposomal Vinblastine Sulfate DaunoXomeVincasar Pfs Decadron Vincristine Delta-Cortef Vinorelbine DeltasoneVinorelbine tartrate Denileukin diftitox VLB DepoCyt VP-16 DexamethasoneVumon Dexamethasone acetate Xeloda dexamethasone sodium Zanosarphosphate Zevalin Dexasone Zinecard Dexrazoxane Zoladex DHAD Zoledronicacid DIC Zometa Diodex Gliadel wafer Docetaxel Glivec Doxil GM-CSFDoxorubicin Goserelin Doxorubicin liposomal granulocyte - colonystimulating factor Droxia Granulocyte macrophage colony stimulatingfactor DTIC Halotestin DTIC-Dome Herceptin Duralone Hexadrol EfudexHexalen Eligard Hexamethylmelamine Ellence HMM Eloxatin Hycamtin ElsparHydrea Emcyt Hydrocort Acetate Epirubicin Hydrocortisone Epoetin alfaHydrocortisone sodium phosphate Erbitux Hydrocortisone sodium succinateErwinia L-asparaginase Hydrocortone phosphate Estramustine HydroxyureaEthyol Ibritumomab Etopophos Ibritumomab Tiuxetan Etoposide IdamycinEtoposide phosphate Idarubicin Eulexin Ifex Evista IFN-alpha ExemestaneIfosfamide Fareston IL-2 Faslodex IL-11 Femara Imatinib mesylateFilgrastim Imidazole Carboxamide Floxuridine Interferon alfa FludaraInterferon Alfa-2b (PEG conjugate) Fludarabine Interleukin-2 FluoroplexInterleukin-11 Fluorouracil Intron A (interferon alfa-2b) Fluorouracil(cream) Leucovorin Fluoxymesterone Leukeran Flutamide Leukine FolinicAcid Leuprolide FUDR Leurocristine Fulvestrant Leustatin G-CSF LiposomalAra-C Gefitinib Liquid Pred Gemcitabine Lomustine Gemtuzumab ozogamicinL-PAM Gemzar L-Sarcolysin Gleevec Meticorten Lupron Mitomycin LupronDepot Mitomycin-C Matulane Mitoxantrone Maxidex M-PrednisolMechlorethamine MTC Mechlorethamine Hydrochlorine MTX MedraloneMustargen Medrol Mustine Megace Mutamycin Megestrol Myleran MegestrolAcetate Iressa Melphalan Irinotecan Mercaptopurine Isotretinoin MesnaKidrolase Mesnex Lanacort Methotrexate L-asparaginase MethotrexateSodium LCR Methylprednisolone Mylocel Letrozole

In one embodiment, the TCN, TCN-P, TCN-PM and/or related compounds andone or more taxanes can be administered together with other cytotoxicagents. In another embodiment, the TCN, TCN-P, TCN-PM and/or relatedcompounds and a one or more taxanes and compositions thereof, when usedin the treatment of solid tumors, can be administered the use ofradiation.

In another embodiment of the present invention, the TCN, TCN-P, TCN-PMand/or related compounds and one or more taxanes and compositionsdisclosed herein can be combined with at least one additionalchemotherapeutic agent. The additional agents can be administered incombination or alternation with the compounds disclosed herein. Thedrugs can form part of the same composition, or be provided as aseparate composition for administration at the same time or a differenttime.

In one embodiment, the TCN, TCN-P, TCN-PM and/or related compounds andone or more taxanes disclosed herein can be combined with antiangiogenicagents to enhance their effectiveness, or combined with otherantiangiogenic agents and administered together with other cytotoxicagents. In another embodiment, the TCN, TCN-P, TCN-PM and/or relatedcompounds and one or more taxanes and compositions, when used in thetreatment of solid tumors, can be administered with the agents selectedfrom, but not limited to IL-12, retinoids, interferons, angiostatin,endostatin, thalidomide, thrombospondin-1, thrombospondin-2, captopryl,anti-neoplastic agents such as alpha interferon, COMP (cyclophosphamide,vincristine, methotrexate and prednisone), etoposide, mBACOD(methortrexate, bleomycin, doxorubicin, cyclophosphamide, vincristineand dexamethasone), PRO-MACE/MOPP (prednisone, methotrexate (w/leucovinrescue), doxorubicin, cyclophosphamide, etoposide/mechlorethamine,vincristine, prednisone and procarbazine), vincristine, vinblastine,angioinhibins, TNP-470, pentosan polysulfate, platelet factor 4,angiostatin, LM-609, SU-101, CM-101, Techgalan, thalidomide, SP-PG andradiation. In further embodiments, the compounds and compositionsdisclosed herein can be administered in combination or alternation with,for example, drugs with antimitotic effects, such as those which targetcytoskeletal elements, including podophyllotoxins or vinca alkaloids(vincristine, vinblastine); antimetabolite drugs (such as5-fluorouracil, cytarabine, gemcitabine, purine analogues such aspentostatin, methotrexate); alkylating agents or nitrogen mustards (suchas nitrosoureas, cyclophosphamide or ifosphamide); drugs which targetDNA such as the anthracycline drugs adriamycin, doxorubicin,pharmorubicin or epirubicin; drugs which target topoisomerases such asetoposide; hormones and hormone agonists or antagonists such asestrogens, antiestrogens (tamoxifen and related compounds) andandrogens, flutamide, leuprorelin, goserelin, cyproterone or octreotide;drugs which target signal transduction in tumour cells includingantibody derivatives such as herceptin; alkylating drugs such asplatinum drugs (cis-platin, carbonplatin, oxaliplatin, paraplatin) ornitrosoureas; drugs potentially affecting metastasis of tumours such asmatrix metalloproteinase inhibitors: gene therapy and antisense agents;antibody therapeutics; other bioactive compounds of marine origin,notably the didemnins such as aplidine; steroid analogues, in particulardexamethasone; anti-inflammatory drugs, including nonsteroidal agents(such as acetaminophen or ibuprofen) or steroids and their derivativesin particular dexamethasone; anti-emetic drugs, including 5HT-3inhibitors (such as gramisetron or ondasetron), and steroids and theirderivatives in particular dexamethasone. In still further embodiments,the compounds and compositions can be used in combination or alternationwith the chemotherapeutic agents disclosed in Table 2.

In one embodiment, the TCN, TCN-P, TCN-PM and/or related compounds and amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, can be administered together with othercytotoxic agents.

In another embodiment, the TCN, TCN-P, TCN-PM and/or related compoundsand a molecule that modulates the HER2/neu (erbB2) receptor, forexample, trastuzumab or a salt thereof, and compositions thereof, whenused in the treatment of solid tumors, can be administered the use ofradiation.

In another embodiment of the present invention, the TCN, TCN-P, TCN-PMand/or related compounds and a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, andcompositions disclosed herein can be combined with at least oneadditional chemotherapeutic agent. The additional agents can beadministered in combination or alternation with the compounds disclosedherein. The drugs can form part of the same composition, or be providedas a separate composition for administration at the same time or adifferent time.

In one embodiment, the TCN, TCN-P, TCN-PM and/or related compounds and amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, disclosed herein can be combined withantiangiogenic agents to enhance their effectiveness, or combined withother antiangiogenic agents and administered together with othercytotoxic agents. In another embodiment, the TCN. TCN-P, TCN-PM and/orrelated compounds and a molecule that modulates the HER2/neu (erbB2)receptor, for example, trastuzumab or a salt thereof, and compositions,when used in the treatment of solid tumors, can be administered with theagents selected from, but not limited to IL-12, retinoids, interferons,angiostatin, endostatin, thalidomide, thrombospondin-1,thrombospondin-2, captopryl, anti-neoplastic agents such as alphainterferon, COMP (cyclophosphamide, vincristine, methotrexate andprednisone), etoposide, mBACOD (methortrexate, bleomycin, doxorubicin,cyclophosphamide, vincristine and dexamethasone), PRO-MACE/MOPP(prednisone, methotrexate (w/leucovin rescue), doxorubicin,cyclophosphamide, etoposide/mechlorethamine, vincristine, prednisone andprocarbazine), vincristine, vinblastine, angioinhibins, TNP-470,pentosan polysulfate, platelet factor 4, angiostatin, LM-609, SU-101,CM-101, Techgalan, thalidomide, SP-PG and radiation. In furtherembodiments, the compounds and compositions disclosed herein can beadministered in combination or alternation with, for example, drugs withantimitotic effects, such as those which target cytoskeletal elements,including podophyllotoxins or vinca alkaloids (vincristine,vinblastine); antimetabolite drugs (such as 5-fluorouracil, cytarabine,gemcitabine, purine analogues such as pentostatin, methotrexate);alkylating agents or nitrogen mustards (such as nitrosoureas,cyclophosphamide or ifosphamide); drugs which target DNA such as theanthracycline drugs adriamycin, doxorubicin, pharmorubicin orepirubicin; drugs which target topoisomerases such as etoposide;hormones and hormone agonists or antagonists such as estrogens,antiestrogens (tamoxifen and related compounds) and androgens,flutamide, leuprorelin, goserelin, cyproterone or octreotide; drugswhich target signal transduction in tumour cells including antibodyderivatives such as herceptin; alkylating drugs such as platinum drugs(cis-platin, carbonplatin, oxaliplatin, paraplatin) or nitrosoureas;drugs potentially affecting metastasis of tumours such as matrixmetalloproteinase inhibitors; gene therapy and antisense agents;antibody therapeutics; other bioactive compounds of marine origin,notably the didemnins such as aplidine; steroid analogues, in particulardexamethasone; anti-inflammatory drugs, including nonsteroidal agents(such as acetaminophen or ibuprofen) or steroids and their derivativesin particular dexamethasone; anti-emetic drugs, including 5HT-3inhibitors (such as gramisetron or ondasetron), and steroids and theirderivatives in particular dexamethasone. In still further embodiments,the compounds and compositions can be used in combination or alternationwith the chemotherapeutic agents disclosed in Table 2.

In one embodiment, the TCN, TCN-P, TCN-PM and/or related compounds andone or more anthracycline compounds of the invention can be administeredtogether with other cytotoxic agents. In another embodiment, the TCN,TCN-P, TCN-PM and/or related compounds and one or more anthracyclinecompounds and compositions thereof, when used in the treatment of solidtumors, can be administered the use of radiation.

In another embodiment of the present invention, the TCN, TCN-P, TCN-PMand/or related compounds and one or more anthracycline compounds andcompositions disclosed herein can be combined with at least oneadditional chemotherapeutic agent. The additional agents can beadministered in combination or alternation with the compounds disclosedherein. The drugs can form part of the same composition, or be providedas a separate composition for administration at the same time or adifferent time.

In one embodiment, the TCN, TCN-P, TCN-PM and/or related compounds andone or more anthracycline compounds disclosed herein can be combinedwith antiangiogenic agents to enhance their effectiveness, or combinedwith other antiangiogenic agents and administered together with othercytotoxic agents. In another embodiment, the TCN, TCN-P, TCN-PM and/orrelated compounds and one or more anthracycline compounds andcompositions, when used in the treatment of solid tumors, can beadministered with the agents selected from, but not limited to IL-12,retinoids, interferons, angiostatin, endostatin, thalidomide,thrombospondin-1, thrombospondin-2, captopryl, anti-neoplastic agentssuch as alpha interferon, COMP (cyclophosphamide, vincristine,methotrexate and prednisone), etoposide, mBACOD (methortrexate,bleomycin, doxorubicin, cyclophosphamide, vincristine anddexamethasone). PRO-MACE/MOPP (prednisone, methotrexate (w/leucovinrescue), doxorubicin, cyclophosphamide, etoposide/mechlorethamine,vincristine, prednisone and procarbazine), vincristine, vinblastine,angioinhibins, TNP-470, pentosan polysulfate, platelet factor 4,angiostatin. LM-609, SU-101, CM-101, Techgalan, thalidomide, SP-PG andradiation. In further embodiments, the compounds and compositionsdisclosed herein can be administered in combination or alternation with,for example, drugs with antimitotic effects, such as those which targetcytoskeletal elements, including podophyllotoxins or vinca alkaloids(vincristine, vinblastine); antimetabolite drugs (such as5-fluorouracil, cytarabine, gemcitabine, purine analogues such aspentostatin, methotrexate); alkylating agents or nitrogen mustards (suchas nitrosoureas, cyclophosphamide or ifosphamide); drugs which targetDNA such as the anthracycline drugs adriamycin, doxorubicin,pharmorubicin or epirubicin; drugs which target topoisomerases such asetoposide: hormones and hormone agonists or antagonists such asestrogens, antiestrogens (tamoxifen and related compounds) andandrogens, flutamide, leuprorelin, goserelin, cyproterone or octreotide;drugs which target signal transduction in tumour cells includingantibody derivatives such as herceptin; alkylating drugs such asplatinum drugs (cis-platin, carbonplatin, oxaliplatin, paraplatin) ornitrosoureas; drugs potentially affecting metastasis of tumours such asmatrix metalloproteinase inhibitors; gene therapy and antisense agents;antibody therapeutics; other bioactive compounds of marine origin,notably the didemnins such as aplidine; steroid analogues, in particulardexamethasone; anti-inflammatory drugs, including nonsteroidal agents(such as acetaminophen or ibuprofen) or steroids and their derivativesin particular dexamethasone; anti-emetic drugs, including 5HT-3inhibitors (such as gramisetron or ondasetron), and steroids and theirderivatives in particular dexamethasone. In still further embodiments,the compounds and compositions can be used in combination or alternationwith the chemotherapeutic agents disclosed in Table 2.

In one embodiment, the TCN, TCN-P, TCN-PM and/or related compounds andan erlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof can be administered together with other cytotoxic agents. Inanother embodiment, the TCN, TCN-P, TCN-PM and/or related compounds andan erlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof and compositions thereof, when used in the treatment of solidtumors, can be administered the use of radiation.

In another embodiment of the invention, the TCN, TCN-P, TCN-PM and/orrelated compounds and an erlotinib-like compound, for example,gefitinib, erlotinib or a salt thereof and compositions disclosed hereincan be combined with at least one additional chemotherapeutic agent. Theadditional agents can be administered in combination or alternation withthe compounds disclosed herein. The drugs can form part of the samecomposition, or be provided as a separate composition for administrationat the same time or a different time.

In one embodiment, the TCN, TCN-P, TCN-PM and/or related compounds andan erlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof disclosed herein can be combined with antiangiogenic agents toenhance their effectiveness, or combined with other antiangiogenicagents and administered together with other cytotoxic agents. In anotherembodiment, the TCN, TCN-P, TCN-PM and/or related compounds and anerlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof and compositions, when used in the treatment of solid tumors,can be administered with the agents selected from, but not limited toIL-12, retinoids, interferons, angiostatin, endostatin, thalidomide,thrombospondin-1, thrombospondin-2, captopryl, anti-neoplastic agentssuch as alpha interferon, COMP (cyclophosphamide, vincristine,methotrexate and prednisone), etoposide, mBACOD (methortrexate,bleomycin, doxorubicin, cyclophosphamide, vincristine anddexamethasone), PRO-MACE/MOPP (prednisone, methotrexate (w/leucovinrescue), doxorubicin, cyclophosphamide, etoposide/mechlorethamine,vincristine, prednisone and procarbazine), vincristine, vinblastine,angioinhibins, TNP-470, pentosan polysulfate, platelet factor 4,angiostatin, LM-609, SU-10, CM-101, Techgalan, thalidomide, SP-PG andradiation. In further embodiments, the compounds and compositionsdisclosed herein can be administered in combination or alternation with,for example, drugs with antimitotic effects, such as those which targetcytoskeletal elements, including podophylotoxins or vinca alkaloids(vincristine, vinblastine); antimetabolite drugs (such as5-fluorouracil, cytarabine, gemcitabine, purine analogues such aspentostatin, methotrexate); alkylating agents or nitrogen mustards (suchas nitrosoureas, cyclophosphamide or ifosphamide); drugs which targetDNA such as the anthracycline drugs adriamycin, doxorubicin,pharmorubicin or epirubicin; drugs which target topoisomerases such asetoposide; hormones and hormone agonists or antagonists such asestrogens, antiestrogens (tamoxifen and related compounds) andandrogens, flutamide, leuprorelin, goserelin, cyproterone or octreotide;drugs which target signal transduction in tumour cells includingantibody derivatives such as herceptin; alkylating drugs such asplatinum drugs (cis-platin, carbonplatin, oxaliplatin, paraplatin) ornitrosoureas; drugs potentially affecting metastasis of tumours such asmatrix metalloproteinase inhibitors; gene therapy and antisense agents;antibody therapeutics; other bioactive compounds of marine origin,notably the didemnins such as aplidine; steroid analogues, in particulardexamethasone; anti-inflammatory drugs, including nonsteroidal agents(such as acetaminophen or ibuprofen) or steroids and their derivativesin particular dexamethasone; anti-emetic drugs, including 5HT-3inhibitors (such as gramisetron or ondasetron), and steroids and theirderivatives in particular dexamethasone. In still further embodiments,the compounds and compositions can be used in combination or alternationwith the chemotherapeutic agents disclosed in Table 2.

In one embodiment, the TCN, TCN-P, TCN-PM and/or related compounds andone or more platinum compounds of the invention can be administeredtogether with other cytotoxic agents. In another embodiment, the TCN,TCN-P, TCN-PM and/or related compounds and one or more platinumcompounds and compositions thereof, when used in the treatment of solidtumors, can be administered the use of radiation.

In another embodiment of the present invention, the TCN, TCN-P, TCN-PMand/or related compounds and one or more platinum compounds andcompositions disclosed herein can be combined with at least oneadditional chemotherapeutic agent. The additional agents can beadministered in combination or alternation with the compounds disclosedherein. The drugs can form part of the same composition, or be providedas a separate composition for administration at the same time or adifferent time.

In one embodiment, the TCN, TCN-P, TCN-PM and/or related compounds andone or more platinum compounds disclosed herein can be combined withantiangiogenic agents to enhance their effectiveness, or combined withother antiangiogenic agents and administered together with othercytotoxic agents. In another embodiment, the TCN, TCN-P, TCN-PM and/orrelated compounds and one or more platinum compounds and compositions,when used in the treatment of solid tumors, can be administered with theagents selected from, but not limited to IL-12, retinoids, interferons,angiostatin, endostatin, thalidomide, thrombospondin-1,thrombospondin-2, captopryl, anti-neoplastic agents such as alphainterferon, COMP (cyclophosphamide, vincristine, methotrexate andprednisone), etoposide, mBACOD (methortrexate, bleomycin, doxorubicin,cyclophosphamide, vincristine and dexamethasone), PRO-MACE/MOPP(prednisone, methotrexate (w/leucovin rescue), doxorubicin,cyclophosphamide, etoposide/mechlorethamine, vincristine, prednisone andprocarbazine), vincristine, vinblastine, angioinhibins, TNP-470,pentosan polysulfate, platelet factor 4, angiostatin, LM-609, SU-101,CM-101, Techgalan, thalidomide, SP-PG and radiation. In furtherembodiments, the compounds and compositions disclosed herein can beadministered in combination or alternation with, for example, drugs withantimitotic effects, such as those which target cytoskeletal elements,including podophylotoxins or vinca alkaloids (vincristine, vinblastine);antimetabolite drugs (such as 5-fluorouracil, cytarabine, gemcitabine,purine analogues such as pentostatin, methotrexate); alkylating agentsor nitrogen mustards (such as nitrosoureas, cyclophosphamide orifosphamide); drugs which target DNA such as the anthracycline drugsadriamycin, doxorubicin, pharmorubicin or epirubicin; drugs which targettopoisomerases such as etoposide; hormones and hormone agonists orantagonists such as estrogens, antiestrogens (tamoxifen and relatedcompounds) and androgens, flutamide, leuprorelin, goserelin, cyproteroneor octreotide; drugs which target signal transduction in tumour cellsincluding antibody derivatives such as herceptin; alkylating drugs suchas platinum drugs (cis-platin, carbonplatin, oxaliplatin, paraplatin) ornitrosoureas; drugs potentially affecting metastasis of tumours such asmatrix metalloproteinase inhibitors; gene therapy and antisense agents;antibody therapeutics; other bioactive compounds of marine origin,notably the didemnins such as aplidine; steroid analogues, in particulardexamethasone; anti-inflammatory drugs, including nonsteroidal agents(such as acetaminophen or ibuprofen) or steroids and their derivativesin particular dexamethasone; anti-emetic drugs, including 5HT-3inhibitors (such as gramisetron or ondasetron), and steroids and theirderivatives in particular dexamethasone. In still further embodiments,the compounds and compositions can be used in combination or alternationwith the chemotherapeutic agents disclosed below in Table 2.

In one embodiment, the TCN, TCN-P, TCN-PM and/or related compounds andbortezomib and derivatives thereof analogs of the invention can beadministered together with other cytotoxic agents. In anotherembodiment, the TCN, TCN-P, TCN-PM and/or related compounds andbortezomib and derivatives thereof analogs and compositions thereof,when used in the treatment of solid tumors, can be administered the useof radiation.

In another embodiment of the present invention, the TCN, TCN-P, TCN-PMand/or related compounds and bortezomib and derivatives thereof analogsand compositions disclosed herein can be combined with at least oneadditional chemotherapeutic agent. The additional agents can beadministered in combination or alternation with the compounds disclosedherein. The drugs can form part of the same composition, or be providedas a separate composition for administration at the same time or adifferent time.

In one embodiment, the TCN, TCN-P, TCN-PM and/or related compounds andbortezomib and derivatives thereof analogs disclosed herein can becombined with antiangiogenic agents to enhance their effectiveness, orcombined with other antiangiogenic agents and administered together withother cytotoxic agents. In another embodiment, the TCN, TCN-P, TCN-PMand/or related compounds and bortezomib and derivatives thereof analogsand compositions, when used in the treatment of solid tumors, can beadministered with the agents selected from, but not limited to IL-12,retinoids, interferons, angiostatin, endostatin, thalidomide,thrombospondin-1, thrombospondin-2, captopryl, anti-neoplastic agentssuch as alpha interferon, COMP (cyclophosphamide, vincristine,methotrexate and prednisone), etoposide, mBACOD (methortrexate,bleomycin, doxorubicin, cyclophosphamide, vincristine anddexamethasone), PRO-MACE/MOPP (prednisone, methotrexate (w/leucovinrescue), doxorubicin, cyclophosphamide, etoposide/mechlorethamine,vincristine, prednisone and procarbazine), vincristine, vinblastine,angioinhibins, TNP-470, pentosan polysulfate, platelet factor 4,angiostatin, LM-609, SU-101, CM-101, Techgalan, thalidomide, SP-PG andradiation. In further embodiments, the compounds and compositionsdisclosed herein can be administered in combination or alternation with,for example, drugs with antimitotic effects, such as those which targetcytoskeletal elements, including podophylotoxins or vinca alkaloids(vincristine, vinblastine); antimetabolite drugs (such as5-fluorouracil, cytarabine, gemcitabine, purine analogues such aspentostatin, methotrexate); alkylating agents or nitrogen mustards (suchas nitrosoureas, cyclophosphamide or ifosphamide); drugs which targetDNA such as the anthracycline drugs adriamycin, doxorubicin,pharmorubicin or epirubicin; drugs which target topoisomerases such asetoposide; hormones and hormone agonists or antagonists such asestrogens, antiestrogens (tamoxifen and related compounds) andandrogens, flutamide, leuprorelin, goserelin, cyproterone or octreotide;drugs which target signal transduction in tumour cells includingantibody derivatives such as herceptin; alkylating drugs such asplatinum drugs (cis-platin, carbonplatin, oxaliplatin, paraplatin) ornitrosoureas; drugs potentially affecting metastasis of tumours such asmatrix metalloproteinase inhibitors; gene therapy and antisense agents;antibody therapeutics; other bioactive compounds of marine origin,notably the didemnins such as aplidine; steroid analogues, in particulardexamethasone; anti-inflammatory drugs, including nonsteroidal agents(such as acetaminophen or ibuprofen) or steroids and their derivativesin particular dexamethasone; anti-emetic drugs, including 5HT-3inhibitors (such as gramisetron or ondasetron), and steroids and theirderivatives in particular dexamethasone. In still further embodiments,the compounds and compositions can be used in combination or alternationwith the chemotherapeutic agents disclosed in Table 2.

In certain embodiments, interferons (IFNs) can be used in combinationswith the compounds of the invention. Suitable interferons include:interferon alpha-2a, interferon alpha-2b, pegylated interferon alpha,including interferon alpha-2a and interferon alpha 2b, interferon beta,interferon gamma, interferon tau, interferon omega, INFERGEN (interferonalphacon-1) by InterMune, OMNIFERON (natural interferon) by Viragen,ALBUFERON by Human Genome Sciences, REBIF (interferon beta-1a) byAres-Serono, Omega Interferon by BioMedicine, Oral Interferon Alpha byAmarillo Biosciences, and interferon gamma, interferon tau, and/orinterferon gamma-1b by InterMune.

In one embodiment TCN, TCN-P, TCN-PM and/or related compounds and one ormore taxanes as disclosed herein can be used in combination oralternation with additional chemotherapeutic agents, such as thosedescribed herein or in Table 2, for the treatment of drug resistantcancer, for example multiple drug resistant cancer. Drug resistantcancers can include cancers of the colon, bone, kidney, adrenal,pancreas, liver and/or any other cancer known in the art or describedherein. In one embodiment, the additional chemotherapeutic agent can bea P-glycoprotein inhibitor. In certain non-limiting embodiments, theP-glycoprotein inhibitor can be selected from the following drugs:verapamil, cyclosporin (such as cyclosporin A), tamoxifen, calmodulinantagonists, dexverapamil, dexniguldipine, valspodar (PSC 833),biricodar (VX-710), tariquidar (XR9576), zosuquidar (LY335979),laniquidar (R101933), and/or ONT-093.

In another embodiment TCN, TCN-P, TCN-PM and/or related compounds and amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, as disclosed herein can be used incombination or alternation with additional chemotherapeutic agents, suchas those described herein or in Table 2, for the treatment of drugresistant cancer, for example multiple drug resistant cancer. Drugresistant cancers can include cancers of the colon, bone, kidney,adrenal, pancreas, liver and/or any other cancer known in the art ordescribed herein. In one embodiment, the additional chemotherapeuticagent can be a P-glycoprotein inhibitor. In certain non-limitingembodiments, the P-glycoprotein inhibitor can be selected from thefollowing drugs: verapamil, cyclosporin (such as cyclosporin A),tamoxifen, calmodulin antagonists, dexverapamil, dexniguldipine,valspodar (PSC 833), biricodar (VX-710), tariquidar (XR9576), zosuquidar(LY335979), laniquidar (R11933), and/or ONT-093.

In another embodiment TCN, TCN-P, TCN-PM and/or related compounds andone or more anthracycline compounds as disclosed herein can be used incombination or alternation with additional chemotherapeutic agents, suchas those described herein or in Table 2, for the treatment of drugresistant cancer, for example multiple drug resistant cancer. Drugresistant cancers can include cancers of the colon, bone, kidney,adrenal, pancreas, liver and/or any other cancer known in the art ordescribed herein. In one embodiment, the additional chemotherapeuticagent can be a P-glycoprotein inhibitor. In certain non-limitingembodiments, the P-glycoprotein inhibitor can be selected from thefollowing drugs: verapamil, cyclosporin (such as cyclosporin A),tamoxifen, calmodulin antagonists, dexverapamil, dexniguldipine,valspodar (PSC 833), biricodar (VX-710), tariquidar (XR9576), zosuquidar(LY335979), laniquidar (R101933), and/or ONT-093.

In another embodiment TCN, TCN-P, TCN-PM and/or related compounds and anerlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof as disclosed herein can be used in combination or alternationwith additional chemotherapeutic agents, such as those described hereinor in Table 2, for the treatment of drug resistant cancer, for examplemultiple drug resistant cancer. Drug resistant cancers can includecancers of the colon, bone, kidney, adrenal, pancreas, liver and/or anyother cancer known in the art or described herein. In one embodiment,the additional chemotherapeutic agent can be a P-glycoprotein inhibitor.In certain non-limiting embodiments, the P-glycoprotein inhibitor can beselected from the following drugs: verapamil, cyclosporin (such ascyclosporin A), tamoxifen, calmodulin antagonists, dexverapamil,dexniguldipine, valspodar (PSC 833), biricodar (VX-710), tariquidar(XR9576), zosuquidar (LY335979), laniquidar (R101933), and/or ONT-093.

In another embodiment TCN, TCN-P, TCN-PM and/or related compounds andone or more platinum compounds as disclosed herein can be used incombination or alternation with additional chemotherapeutic agents, suchas those described herein or in Table 2, for the treatment of drugresistant cancer, for example multiple drug resistant cancer. Drugresistant cancers can include cancers of the colon, bone, kidney,adrenal, pancreas, liver and/or any other cancer known in the art ordescribed herein. In one embodiment, the additional chemotherapeuticagent can be a P-glycoprotein inhibitor. In certain non-limitingembodiments, the P-glycoprotein inhibitor can be selected from thefollowing drugs: verapamil, cyclosporin (such as cyclosporin A),tamoxifen, calmodulin antagonists, dexverapamil, dexniguldipine,valspodar (PSC 833), biricodar (VX-710), tariquidar (XR9576), zosuquidar(LY335979), laniquidar (R101933), and/or ONT-093.

In another embodiment TCN, TCN-P, TCN-PM and/or related compounds andbortezomib or a salt or derivative thereof as disclosed herein can beused in combination or alternation with additional chemotherapeuticagents, such as those described herein or in Table 2, for the treatmentof drug resistant cancer, for example multiple drug resistant cancer.Drug resistant cancers can include cancers of the colon, bone, kidney,adrenal, pancreas, liver and/or any other cancer known in the art ordescribed herein. In one embodiment, the additional chemotherapeuticagent can be a P-glycoprotein inhibitor. In certain non-limitingembodiments, the P-glycoprotein inhibitor can be selected from thefollowing drugs: verapamil, cyclosporin (such as cyclosporin A),tamoxifen, calmodulin antagonists, dexverapamil, dexniguldipine,valspodar (PSC 833), biricodar (VX-710), tariquidar (XR9576), zosuquidar(LY335979), laniquidar (R101933), and/or ONT-093.

In one embodiment TCN, TCN-P, TCN-PM and/or related compounds and one ormore taxanes as disclosed herein can be used in combination oralternation with additional chemotherapeutic agents, such as thosedescribed herein or in Table 2, for the treatment of drug resistantcancer, for example multiple drug resistant cancer.

In one embodiment TCN, TCN-P, TCN-PM and/or related compounds and amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof as disclosed herein can be used incombination or alternation with additional chemotherapeutic agents, suchas those described herein or in Table 2, for the treatment of drugresistant cancer, for example multiple drug resistant cancer.

In one embodiment TCN, TCN-P, TCN-PM and/or related compounds andanthracycline compounds as disclosed herein can be used in combinationor alternation with additional chemotherapeutic agents, such as thosedescribed herein or in Table 2, for the treatment of drug resistantcancer, for example multiple drug resistant cancer.

In one embodiment TCN, TCN-P, TCN-PM and/or related compounds and anerlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof as disclosed herein can be used in combination or alternationwith additional chemotherapeutic agents, such as those described hereinor in Table 2, for the treatment of drug resistant cancer, for examplemultiple drug resistant cancer.

In one embodiment TCN, TCN-P, TCN-PM and/or related compounds and one ormore platinum compounds as disclosed herein can be used in combinationor alternation with additional chemotherapeutic agents, such as thosedescribed herein or in Table 2, for the treatment of drug resistantcancer, for example multiple drug resistant cancer.

In one embodiment TCN, TCN-P, TCN-PM and/or related compounds andbortezomib or a salt or derivative thereof as disclosed herein can beused in combination or alternation with additional chemotherapeuticagents, such as those described herein or in Table 2, for the treatmentof drug resistant cancer, for example multiple drug resistant cancer.

7. PHARMACEUTICAL COMPOSITIONS

The compositions including TCN, TCN-P, TCN-PM and/or related compoundsand one or more taxanes can optionally be administered with apharmaceutical carrier or excipient. Pharmaceutical carriers suitablefor administration of the compounds provided herein include any suchcarriers known to those skilled in the art to be suitable for theparticular mode of administration. The TCN, TCN-P, TCN-PM and/or relatedcompounds and in combination with one or more taxanes may be formulatedas the sole pharmaceutically active ingredient in the composition or maybe combined with one or more taxanes.

Compositions including the TCN, TCN-P, TCN-PM and/or related compoundsand one or more taxanes may be suitable for oral, rectal, nasal, topical(including buccal and sublingual), vaginal, or parenteral (includingsubcutaneous, intramuscular, subcutaneous, intravenous, intradermal,intraocular, intratracheal, intracisternal, intraperitoneal, andepidural) administration. Preferably the compositions are administeredintravenously.

The compositions including TCN, TCN-P, TCN-PM and/or related compoundsand a molecule that modulates the HER2/neu (erbB2) receptor, forexample, trastuzumab or a salt thereof, can optionally be administeredwith a pharmaceutical carrier or excipient. Pharmaceutical carrierssuitable for administration of the compounds provided herein include anysuch carriers known to those skilled in the art to be suitable for theparticular mode of administration. The TCN, TCN-P, TCN-PM and/or relatedcompounds and in combination with a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, may beformulated as the sole pharmaceutically active ingredients in thecomposition or may be combined.

Compositions including the TCN, TCN-P, TCN-PM and/or related compoundsand a molecule that modulates the HER2/neu (erbB2) receptor, forexample, trastuzumab or a salt thereof, may be suitable for oral,rectal, nasal, topical (including buccal and sublingual), vaginal, orparenteral (including subcutaneous, intramuscular, subcutaneous,intravenous, intradermal, intraocular, intratracheal, intracisternal,intraperitoneal, and epidural) administration. Preferably thecompositions are administered intravenously.

The compositions including TCN, TCN-P, TCN-PM and/or related compoundsand one or more anthracycline analogs can optionally be administeredwith a pharmaceutical carrier or excipient. Pharmaceutical carrierssuitable for administration of the compounds provided herein include anysuch carriers known to those skilled in the art to be suitable for theparticular mode of administration. The TCN, TCN-P, TCN-PM and/or relatedcompounds and in combination with one or more anthracycline analogs maybe formulated as the sole pharmaceutically active ingredient in thecomposition or may be combined with one or more anthracycline analogs.

Compositions including the TCN, TCN-P, TCN-PM and/or related compoundsand one or more anthracycline analogs may be suitable for oral, rectal,nasal, topical (including buccal and sublingual), vaginal, or parenteral(including subcutaneous, intramuscular, subcutaneous, intravenous,intradermal, intraocular, intratracheal, intracisternal,intraperitoneal, and epidural) administration. Preferably thecompositions are administered intravenously.

The compositions including TCN, TCN-P, TCN-PM and/or related compoundsand an erlotinib-like compound, for example, gefitinib, erlotinib or asalt thereof can optionally be administered with a pharmaceuticalcarrier or excipient. Pharmaceutical carriers suitable foradministration of the compounds provided herein include any suchcarriers known to those skilled in the art to be suitable for theparticular mode of administration. The TCN, TCN-P, TCN-PM and/or relatedcompounds and in combination with an erlotinib-like compound, forexample, gefitinib, erlotinib or a salt thereof may be formulated as thesole pharmaceutically active ingredients in the composition or may becombined.

Compositions including the TCN, TCN-P, TCN-PM and/or related compoundsand an erlotinib-like compound, for example, gefitinib, erlotinib or asalt thereof may be suitable for oral, rectal, nasal, topical (includingbuccal and sublingual), vaginal, or parenteral (including subcutaneous,intramuscular, subcutaneous, intravenous, intradermal, intraocular,intratracheal, intracisternal, intraperitoneal, and epidural)administration. Preferably the compositions are administeredintravenously.

The compositions including TCN, TCN-P, TCN-PM and/or related compoundsand one or more platinum compounds can optionally be administered with apharmaceutical carrier or excipient. Pharmaceutical carriers suitablefor administration of the compounds provided herein include any suchcarriers known to those skilled in the art to be suitable for theparticular mode of administration. The TCN, TCN-P, TCN-PM and/or relatedcompounds and in combination with one or more platinum compounds may beformulated as the sole pharmaceutically active ingredient in thecomposition or may be combined with one or more platinum compounds.

Compositions including the TCN, TCN-P, TCN-PM and/or related compoundsand one or more platinum compounds may be suitable for oral, rectal,nasal, topical (including buccal and sublingual), vaginal, or parenteral(including subcutaneous, intramuscular, subcutaneous, intravenous,intradermal, intraocular, intratracheal, intracisternal,intraperitoneal, and epidural) administration. Preferably thecompositions are administered intravenously.

The compositions including TCN, TCN-P, TCN-PM and/or related compoundsand bortezomib and derivatives thereof analogs can optionally beadministered with a pharmaceutical carrier or excipient. Pharmaceuticalcarriers suitable for administration of the compounds provided hereininclude any such carriers known to those skilled in the art to besuitable for the particular mode of administration. The TCN, TCN-P,TCN-PM and/or related compounds and in combination with bortezomib andderivatives thereof analogs may be formulated as the solepharmaceutically active ingredient in the composition or may be combinedwith bortezomib and derivatives thereof analogs.

Compositions including the TCN, TCN-P, TCN-PM and/or related compoundsand bortezomib and derivatives thereof analogs may be suitable for oral,rectal, nasal, topical (including buccal and sublingual), vaginal, orparenteral (including subcutaneous, intramuscular, subcutaneous,intravenous, intradermal, intraocular, intratracheal, intracisternal,intraperitoneal, and epidural) administration. Preferably thecompositions are administered intravenously.

The compositions may conveniently be presented in unit dosage form andmay be prepared by conventional pharmaceutical techniques. Suchtechniques include the step of bringing into association one or morecompositions of the present invention and one or more pharmaceuticalcarriers or excipients.

The TCN, TCN-P, TCN-PM and/or related compounds and one or more taxanesand compositions thereof can be formulated into suitable pharmaceuticalpreparations such as solutions, suspensions, tablets, dispersibletablets, pills, capsules, powders, sustained release formulations orelixirs, for oral administration or in sterile solutions or suspensionsfor parenteral administration, as well as transdermal patch preparationand dry powder inhalers. In one embodiment, the TCN, TCN-P, TCN-PMand/or related compounds described above are formulated intopharmaceutical compositions using techniques and procedures well knownin the art (see, e.g., Ansel Introduction to Pharmaceutical DosageForms, Fourth Edition 1985, 126).

The TCN, TCN-P, TCN-PM and/or related compounds and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, and compositions thereof can be formulated into suitablepharmaceutical preparations such as solutions, suspensions, tablets,dispersible tablets, pills, capsules, powders, sustained releaseformulations or elixirs, for oral administration or in sterile solutionsor suspensions for parenteral administration, as well as transdermalpatch preparation and dry powder inhalers. In one embodiment, the TCN,TCN-P, TCN-PM and/or related compounds described above are formulatedinto pharmaceutical compositions using techniques and procedures wellknown in the art (see e.g., Ansel, Introduction to Pharmaceutical DosageForms, Fourth Ed., 1985, p. 126).

The TCN, TCN-P, TCN-PM and/or related compounds and one or moreanthracycline analogs and compositions thereof can be formulated intosuitable pharmaceutical preparations such as solutions, suspensions,tablets, dispersible tablets, pills, capsules, powders, sustainedrelease formulations or elixirs, for oral administration or in sterilesolutions or suspensions for parenteral administration, as well astransdermal patch preparation and dry powder inhalers. In oneembodiment, the TCN, TCN-P, TCN-PM and/or related compounds describedabove are formulated into pharmaceutical compositions using techniquesand procedures well known in the art (see, e.g., Ansel Introduction toPharmaceutical Dosage Forms, Fourth Edition 1985, 126).

The TCN, TCN-P, TCN-PM and/or related compounds and an erlotinib-likecompound, for example, gefitinib, erlotinib or a salt thereof andcompositions thereof can be formulated into suitable pharmaceuticalpreparations such as solutions, suspensions, tablets, dispersibletablets, pills, capsules, powders, sustained release formulations orelixirs, for oral administration or in sterile solutions or suspensionsfor parenteral administration, as well as transdermal patch preparationand dry powder inhalers. In one embodiment, the TCN, TCN-P, TCN-PMand/or related compounds described above are formulated intopharmaceutical compositions using techniques and procedures well knownin the art (see, e.g., Ansel, Introduction to Pharmaceutical DosageForms, Fourth Ed., 1985, p. 126).

The TCN, TCN-P, TCN-PM and/or related compounds and one or more platinumcompounds and compositions thereof can be formulated into suitablepharmaceutical preparations such as solutions, suspensions, tablets,dispersible tablets, pills, capsules, powders, sustained releaseformulations or elixirs, for oral administration or in sterile solutionsor suspensions for parenteral administration, as well as transdermalpatch preparation and dry powder inhalers. In one embodiment, the TCN,TCN-P, TCN-PM and/or related compounds described above are formulatedinto pharmaceutical compositions using techniques and procedures wellknown in the art (see, e.g., Ansel Introduction to Pharmaceutical DosageForms, Fourth Ed., 1985, p. 126).

The TCN, TCN-P, TCN-PM and/or related compounds and bortezomib andderivatives thereof analogs and compositions thereof can be formulatedinto suitable pharmaceutical preparations such as solutions,suspensions, tablets, dispersible tablets, pills, capsules, powders,sustained release formulations or elixirs, for oral administration or insterile solutions or suspensions for parenteral administration, as wellas transdermal patch preparation and dry powder inhalers. In oneembodiment, the TCN, TCN-P, TCN-PM and/or related compounds describedabove are formulated into pharmaceutical compositions using techniquesand procedures well known in the art (see, e.g., Ansel Introduction toPharmaceutical Dosage Forms, Fourth Edition 1985, 126).

In the compositions, effective concentrations of one or more compoundsor pharmaceutically acceptable derivatives thereof may be mixed with oneor more suitable pharmaceutical carriers. The compounds of the inventionmay be derivatized as the corresponding salts, esters, enol ethers oresters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids,bases, solvates, hydrates or prodrugs prior to formulation. Theconcentrations of the compounds in the compositions are effective fordelivery of an amount, upon administration, that treats, prevents, orameliorates one or more of the symptoms of the target disease ordisorder. In one embodiment, the compositions are formulated for singledosage administration. To formulate a composition, the weight fractionof compound is dissolved, suspended, dispersed or otherwise mixed in aselected carrier at an effective concentration such that the treatedcondition is relieved, prevented, or one or more symptoms areameliorated.

Compositions suitable for oral administration may be presented asdiscrete units such as, but not limited to, tablets, caplets, pills ordragees capsules, or cachets, each containing a predetermined amount ofone or more of the compositions; as a powder or granules; as a solutionor a suspension in an aqueous liquid or a non-aqueous liquid; or as anoil-in-water liquid emulsion or a water-in-oil emulsion or as a bolus,etc.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing a TCN, TCN-P,TCN-PM and/or related compounds and optional pharmaceutical adjuvants ina carrier, such as, for example, water, saline, aqueous dextrose,glycerol, glycols, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting agents, emulsifying agents, solubilizingagents, pH buffering agents, preservatives, flavoring agents, and thelike, for example, acetate, sodium citrate. cyclodextrine derivatives,sorbitan monolaurate, triethanolamine sodium acetate, triethanolamineoleate, and other such agents. Methods of preparing such dosage formsare known, or will be apparent, to those skilled in this art; forexample, see Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa., 15th Edition, 1975.

Compositions of the present invention suitable for topicaladministration in the mouth include for example, lozenges, having theingredients in a flavored basis, usually sucrose and acacia ortragacanth; pastilles, having one or more TCN, TCN-P, TCN-PM and/orrelated compounds and one or more taxanes of the present invention in aninert basis such as gelatin and glycerin, or sucrose and acacia; andmouthwashes, having one or more of the compositions of the presentinvention administered in a suitable liquid carrier.

Compositions of the present invention suitable for topicaladministration in the mouth include for example, lozenges, having theingredients in a flavored basis, usually sucrose and acacia ortragacanth; pastilles, having one or more TCN, TCN-P, TCN-PM and/orrelated compounds and a molecule that modulates the HER2/neu (erbB2)receptor, for example, trastuzumab or a salt thereof, of the presentinvention in an inert basis such as gelatin and glycerin, or sucrose andacacia; and mouthwashes, having one or more of the compositions of thepresent invention administered in a suitable liquid carrier.

Compositions of the present invention suitable for topicaladministration in the mouth include for example, lozenges, having theingredients in a flavored basis, usually sucrose and acacia ortragacanth; pastilles, having one or more TCN, TCN-P, TCN-PM and/orrelated compounds and one or more anthracycline analogs of the presentinvention in an inert basis such as gelatin and glycerin, or sucrose andacacia; and mouthwashes, having one or more of the compositions of thepresent invention administered in a suitable liquid carrier.

Compositions of the present invention suitable for topicaladministration in the mouth include for example, lozenges, having theingredients in a flavored basis, usually sucrose and acacia ortragacanth; pastilles, having one or more TCN, TCN-P, TCN-PM and/orrelated compounds and an erlotinib-like compound, for example,gefitinib, erlotinib or a salt thereof of the present invention in aninert basis such as gelatin and glycerin, or sucrose and acacia; andmouthwashes, having one or more of the compositions of the presentinvention administered in a suitable liquid carrier.

Compositions of the present invention suitable for topicaladministration in the mouth include for example, lozenges, having theingredients in a flavored basis, usually sucrose and acacia ortragacanth; pastilles, having one or more TCN, TCN-P, TCN-PM and/orrelated compounds and one or more platinum compounds of the presentinvention in an inert basis such as gelatin and glycerin, or sucrose andacacia; and mouthwashes, having one or more of the compositions of thepresent invention administered in a suitable liquid carrier.

Compositions of the present invention suitable for topicaladministration in the mouth include for example, lozenges, having theingredients in a flavored basis, usually sucrose and acacia ortragacanth; pastilles, having TCN, TCN-P, TCN-PM and/or relatedcompounds and bortezomib and derivatives thereof analogs of the presentinvention in an inert basis such as gelatin and glycerin, or sucrose andacacia; and mouthwashes, having of the compositions of the presentinvention administered in a suitable liquid carrier.

The tablets, pills, capsules, troches and the like can contain one ormore of the following ingredients, or compounds of a similar nature: abinder; a lubricant; a diluent; a glidant; a disintegrating agent; acoloring agent; a sweetening agent; a flavoring agent; a wetting agent;an emetic coating; and a film coating. Examples of binders includemicrocrystalline cellulose, gum tragacanth, glucose solution, acaciamucilage, gelatin solution, molasses, polyinylpyrrolidine, povidone,crospovidones, sucrose and starch paste. Lubricants include talc,starch, magnesium or calcium stearate, lycopodium and stearic acid.Diluents include, for example, lactose, sucrose, starch, kaolin, salt,mannitol and dicalcium phosphate. Glidants include, but are not limitedto, colloidal silicon dioxide. Disintegrating agents includecroscarmellose sodium, sodium starch glycolate, alginic acid, cornstarch, potato starch, bentonite, methylcellulose, agar andcarboxymethylcellulose. Coloring agents include, for example, any of theapproved certified water soluble FD and C dyes, mixtures thereof; andwater insoluble FD and C dyes suspended on alumina hydrate. Sweeteningagents include sucrose, lactose, mannitol and artificial sweeteningagents such as saccharin, and any number of spray dried flavors.Flavoring agents include natural flavors extracted from plants such asfruits and synthetic blends of compounds which produce a pleasantsensation, such as, but not limited to peppermint and methyl salicylate.Wetting agents include propylene glycol monostearate, sorbitanmonooleate, diethylene glycol monolaurate and polyoxyethylene lauralether. Emetic-coatings include fatty acids, fats, waxes, shellac,ammoniated shellac and cellulose acetate phthalates. Film coatingsinclude hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate.

Compositions suitable for topical administration to the skin may bepresented as ointments, creams, gels, and pastes, having one or more ofthe compositions administered in a pharmaceutical acceptable carrier.

Compositions for rectal administration may be presented as a suppositorywith a suitable base including, for example, cocoa butter or asalicylate.

Compositions suitable for nasal administration, when the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of 20 to 500 microns which is administered in the manner inwhich snuff is taken, (i.e., by rapid inhalation through the nasalpassage from a container of the powder held close up to the nose). Whenthe carrier is a liquid (for example, a nasal spray or as nasal drops),one or more of the compositions can be admixed in an aqueous or oilysolution, and inhaled or sprayed into the nasal passage.

Compositions suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining one or more of the compositions and appropriate carriers.

Compositions suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats, and solutes which render the formulationisotonic with the blood of the intended recipient; and aqueous andnon-aqueous sterile suspensions which may include suspending agents andthickening agents. The compositions may be presented in unit-dose ormulti-dose containers, for example, sealed ampules and vials, and may bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules, andtablets of the kind previously described above.

Pharmaceutical organic or inorganic solid or liquid carrier mediasuitable for enteral or parenteral administration can be used tofabricate the compositions. Gelatin, lactose, starch, magnesiumstearate, talc, vegetable and animal fats and oils, gum, polyalkyleneglycol, water, or other known carriers may all be suitable as carriermedia.

Compositions including TCN, TCN-P, TCN-PM and/or related compounds andone or more taxanes may be used in combination with one or morepharmaceutically acceptable carrier mediums and/or excipients. As usedherein, “pharmaceutically acceptable carrier medium” includes any andall carriers, solvents, diluents, or other liquid vehicles, dispersionor suspension aids, surface active agents, isotonic agents, thickeningor emulsifying agents, preservatives, solid binders, lubricants,adjuvants, vehicles, delivery systems, disintegrants, absorbents,preservatives, surfactants, colorants, flavorants, or sweeteners and thelike, as suited to the particular dosage form desired.

Additionally, the compositions including TCN, TCN-P, TCN-PM and/orrelated compounds and one or more taxanes may be combined withpharmaceutically acceptable excipients, and, optionally,sustained-release matrices, such as biodegradable polymers, to formtherapeutic compositions. A “pharmaceutically acceptable excipient”includes a non-toxic solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type.

Compositions including TCN, TCN-P, TCN-PM and/or related compounds and amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof; may be used in combination with one ormore pharmaceutically acceptable carrier mediums and/or excipients. Asused herein, “pharmaceutically acceptable carrier medium” includes anyand all carriers, solvents, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants, adjuvants, vehicles, delivery systems, disintegrants,absorbents, preservatives, surfactants, colorants, flavorants, orsweeteners and the like, as suited to the particular dosage formdesired.

Additionally, the compositions including TCN, TCN-P, TCN-PM and/orrelated compounds and a molecule that modulates the HER2/neu (erbB2)receptor, for example, trastuzumab or a salt thereof, may be combinedwith pharmaceutically acceptable excipients, and, optionally,sustained-release matrices, such as biodegradable polymers, to formtherapeutic compositions. A “pharmaceutically acceptable excipient”includes a non-toxic solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type.

Compositions including TCN, TCN-P, TCN-PM and/or related compounds andone or more anthracycline analogs may be used in combination with one ormore pharmaceutically acceptable carrier mediums and/or excipients. Asused herein, “pharmaceutically acceptable carrier medium” includes anyand all carriers, solvents, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants, adjuvants, vehicles, delivery systems, disintegrants,absorbents, preservatives, surfactants, colorants, flavorants, orsweeteners and the like, as suited to the particular dosage formdesired.

Additionally, the compositions including TCN, TCN-P, TCN-PM and/orrelated compounds and one or more anthracycline analogs may be combinedwith pharmaceutically acceptable excipients, and, optionally,sustained-release matrices, such as biodegradable polymers, to formtherapeutic compositions. A “pharmaceutically acceptable excipient”includes a non-toxic solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type.

Compositions including TCN, TCN-P, TCN-PM and/or related compounds andan erlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof may be used in combination with one or more pharmaceuticallyacceptable carrier mediums and/or excipients. As used herein,“pharmaceutically acceptable carrier medium” includes any and allcarriers, solvents, diluents, or other liquid vehicles, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants, adjuvants,vehicles, delivery systems, disintegrants, absorbents, preservatives,surfactants, colorants, flavorants, or sweeteners and the like, assuited to the particular dosage form desired.

Additionally, the compositions including TCN, TCN-P, TCN-PM and/orrelated compounds and an erlotinib-like compound, for example,gefitinib, erlotinib or a salt thereof may be combined withpharmaceutically acceptable excipients, and, optionally,sustained-release matrices, such as biodegradable polymers, to formtherapeutic compositions. A “pharmaceutically acceptable excipient”includes a non-toxic solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type.

Compositions including TCN, TCN-P, TCN-PM and/or related compounds andone or more platinum compounds may be used in combination with one ormore pharmaceutically acceptable carrier mediums and/or excipients. Asused herein, “pharmaceutically acceptable carrier medium” includes anyand all carriers, solvents, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants, adjuvants, vehicles, delivery systems, disintegrants,absorbents, preservatives, surfactants, colorants, flavorants, orsweeteners and the like, as suited to the particular dosage formdesired.

Additionally, the compositions including TCN, TCN-P, TCN-PM and/orrelated compounds and one or more platinum compounds may be combinedwith pharmaceutically acceptable excipients, and, optionally,sustained-release matrices, such as biodegradable polymers, to formtherapeutic compositions. A “pharmaceutically acceptable excipient”includes a non-toxic solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type.

Compositions including TCN, TCN-P, TCN-PPM and/or related compounds andbortezomib and derivatives thereof analogs may be used in combinationwith pharmaceutically acceptable carrier mediums and/or excipients. Asused herein, “pharmaceutically acceptable carrier medium” includes anyand all carriers, solvents, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants, adjuvants, vehicles, delivery systems, disintegrants,absorbents, preservatives, surfactants, colorants, flavorants, orsweeteners and the like, as suited to the particular dosage formdesired.

Additionally, the compositions including TCN, TCN-P, TCN-PM and/orrelated compounds and bortezomib and derivatives thereof analogs may becombined with pharmaceutically acceptable excipients, and, optionally,sustained-release matrices, such as biodegradable polymers, to formtherapeutic compositions. A “pharmaceutically acceptable excipient”includes a non-toxic solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type.

It will be understood, however, that the total daily usage of thecompositions will be decided by the attending physician within the scopeof sound medical judgment. The specific therapeutically effective doselevel for any particular host will depend upon a variety of factors,including for example, the disorder being treated and the severity ofthe disorder; activity of the specific composition employed; thespecific composition employed, the age, body weight, general health, sexand diet of the patient; the time of administration; route ofadministration; rate of excretion of the specific compound employed; theduration of the treatment; the TCN, TCN-P, TCN-PM and/or relatedcompounds and/or the taxane used in combination or coincidental with thespecific composition employed; the TCN, TCN-P, TCN-PM and/or relatedcompounds and one or more molecules that modulate the HER2/neu (erbB2)receptor, for example, trastuzumab or a salt thereof used in combinationor coincidental with the specific composition employed; the TCN, TCN-P,TCN-PM and/or related compounds and/or the anthracycline analogs used incombination or coincidental with the specific composition employed; anerlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof used in combination or coincidental with the specificcomposition employed; the TCN, TCN-P, TCN-PM and/or related compoundsand/or one or more platinum compounds used in combination orcoincidental with the specific composition employed, the TCN, TCN-P,TCN-PM and/or related compounds and/or one or more bortezomib andderivatives thereof used in combination or coincidental with thespecific composition employed, and like factors well known in themedical arts. For example, it is well within the skill of the art tostart doses of the composition at levels lower than those required toachieve the desired therapeutic effect and to gradually increase thedosage until the desired effect is achieved.

Compositions including TCN, TCN-P, TCN-PM and/or related compounds andone or more taxanes are preferably formulated in dosage unit form forease of administration and uniformity of dosage. “Dosage unit form” asused herein refers to a physically discrete unit of the compositionappropriate for the host to be treated. Each dosage should contain thequantity of composition calculated to produce the desired therapeuticaffect either as such, or in association with the selectedpharmaceutical carrier medium.

Compositions including TCN, TCN-P, TCN-PM and/or related compounds and amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, are preferably formulated in dosage unitform for ease of administration and uniformity of dosage. “Dosage unitform” as used herein refers to a physically discrete unit of thecomposition appropriate for the host to be treated. Each dosage shouldcontain the quantity of composition calculated to produce the desiredtherapeutic affect either as such, or in association with the selectedpharmaceutical carrier medium.

Compositions including TCN, TCN-P, TCN-PM and/or related compounds andone or more anthracycline analogs are preferably formulated in dosageunit form for ease of administration and uniformity of dosage. “Dosageunit form” as used herein refers to a physically discrete unit of thecomposition appropriate for the host to be treated. Each dosage shouldcontain the quantity of composition calculated to produce the desiredtherapeutic affect either as such, or in association with the selectedpharmaceutical carrier medium.

Compositions including TCN, TCN-P, TCN-PM and/or related compounds andan erlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof are preferably formulated in dosage unit form for ease ofadministration and uniformity of dosage. “Dosage unit form” as usedherein refers to a physically discrete unit of the compositionappropriate for the host to be treated. Each dosage should contain thequantity of composition calculated to produce the desired therapeuticaffect either as such, or in association with the selectedpharmaceutical carrier medium.

Compositions including TCN, TCN-P, TCN-PM and/or related compounds andone or more platinum compounds are preferably formulated in dosage unitform for ease of administration and uniformity of dosage. “Dosage unitform” as used herein refers to a physically discrete unit of thecomposition appropriate for the host to be treated. Each dosage shouldcontain the quantity of composition calculated to produce the desiredtherapeutic affect either as such, or in association with the selectedpharmaceutical carrier medium.

Compositions including TCN, TCN-P, TCN-PM and/or related compounds andbortezomib and derivatives thereof analogs are preferably formulated indosage unit form for ease of administration and uniformity of dosage.“Dosage unit form” as used herein refers to a physically discrete unitof the composition appropriate for the host to be treated. Each dosageshould contain the quantity of composition calculated to produce thedesired therapeutic affect either as such, or in association with theselected pharmaceutical carrier medium.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, or an appropriate fraction thereof, of theadministered ingredient. For example, approximately 1-50 mg per day of acompound disclosed herein can reduce the volume of a solid tumor inmice.

The dosage will depend on host factors such as weight, age, surfacearea, metabolism, tissue distribution, absorption rate and excretionrate. In one embodiment, approximately 0.5 to 7 grams per day of a TCN,TCN-P, TCN-PM and/or related compounds disclosed herein may beadministered to humans. Optionally, approximately 1 to 4 grams per dayof the compound can be administered to humans. In certain embodiments0.001-5 mg/day is administered to a human. The therapeutically effectivedose level will depend on many factors as noted above. In addition, itis well within the skill of the art to start doses of the composition atrelatively low levels, and increase the dosage until the desired effectis achieved.

Compositions including TCN, TCN-P, TCN-PM and/or related compounds andone or more taxanes may be used with a sustained-release matrix, whichcan be made of materials, usually polymers, which are degradable byenzymatic or acid-based hydrolysis or by dissolution. Once inserted intothe body, the matrix is acted upon by enzymes and body fluids. Asustained-release matrix for example is chosen from biocompatiblematerials such as liposomes, polylactides (polylactic acid),polyglycolide (polymer of glycolic acid), polylactide co-glycolide(copolymers of lactic acid and glycolic acid), polyanhydrides,poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitinsulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides,nucleic acids, polyamino acids, amino acids such as phenylalanine,tyrosine, isoleucine, polynucleotides, polyvinyl propylene,polyvinylpyrrolidone and silicone. A preferred biodegradable matrix is amatrix of one of either polylactide, polyglycolide, or polylactideco-glycolide (co-polymers of lactic acid and glycolic acid).

The TCN, TCN-P, TCN-PM and/or related compounds and one or more taxanesmay also be administered in the form of liposomes. As is known in theart, liposomes are generally derived from phospholipids or other lipidsubstances. Liposomes are formed by mono- or multi-lamellar hydratedliquid crystals that are dispersed in an aqueous medium. Any non-toxic,physiologically-acceptable and metabolizable lipid capable of formingliposomes can be used. The liposome can contain, in addition to one ormore compositions of the present invention, stabilizers, preservatives,excipients, and the like. Examples of lipids are the phospholipids andthe phosphatidyl cholines (lecithins), both natural and synthetic.Methods to form liposomes are known in the art.

The TCN, TCN-P, TCN-PM and/or related compounds and one or more taxanesmay be formulated as aerosols for application, such as by inhalation.These formulations for administration to the respiratory tract can be inthe form of an aerosol or solution for a nebulizer, or as a microfinepowder for insufflation, alone or in combination with an inert carriersuch as lactose. In such a case, the particles of the formulation will,in one embodiment, have diameters of less than 50 microns, in oneembodiment less than 10 microns.

Compositions including the TCN, TCN-P, TCN-PM and/or related compoundsand one or more taxanes may be used in combination with othercompositions and/or procedures for the treatment of the conditionsdescribed above. For example, a tumor may be treated conventionally withsurgery, radiation, or chemotherapy combined with one or morecompositions of the present invention and then one or more compositionsof the present invention may be subsequently administered to the patientto extend the dormancy of micrometastases and to stabilize, inhibit, orreduce the growth of any residual primary tumor.

Compositions including TCN, TCN-P, TCN-PM and/or related compounds and amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, may be used with a sustained-releasematrix, which can be made of materials, usually polymers, which aredegradable by enzymatic or acid-based hydrolysis or by dissolution. Onceinserted into the body, the matrix is acted upon by enzymes and bodyfluids. A sustained-release matrix for example is chosen frombiocompatible materials such as liposomes, polylactides (polylacticacid), polyglycolide (polymer of glycolic acid), polylactideco-glycolide (copolymers of lactic acid and glycolic acid),polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid,collagen, chondroitin sulfate, carboxylic acids, fatty acids,phospholipids, polysaccharides, nucleic acids, polyamino acids, aminoacids such as phenylalanine, tyrosine, isoleucine, polynucleotides,polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferredbiodegradable matrix is a matrix of one of either polylactide,polyglycolide, or polylactide co-glycolide (co-polymers of lactic acidand glycolic acid).

The TCN, TCN-P, TCN-PM and/or related compounds and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, may also be administered in the form of liposomes. As isknown in the art, liposomes are generally derived from phospholipids orother lipid substances. Liposomes are formed by mono- or multi-lamellarhydrated liquid crystals that are dispersed in an aqueous medium. Anynon-toxic, physiologically-acceptable and metabolizable lipid capable offorming liposomes can be used. The liposome can contain, in addition toone or more compositions of the present invention, stabilizers,preservatives, excipients, and the like. Examples of lipids are thephospholipids and the phosphatidyl cholines (lecithins), both naturaland synthetic. Methods to form liposomes are known in the art.

The TCN, TCN-P, TCN-PM and/or related compounds and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, may be formulated as aerosols for application, such as byinhalation. These formulations for administration to the respiratorytract can be in the form of an aerosol or solution for a nebulizer, oras a microfine powder for insufflation, alone or in combination with aninert carrier such as lactose. In such a case, the particles of theformulation will, in one embodiment, have diameters of less than 50microns, in one embodiment less than 10 microns.

Compositions including the TCN, TCN-P, TCN-PM and/or related compoundsand a molecule that modulates the HER2/neu (erbB2) receptor, forexample, trastuzumab or a salt thereof, may be used in combination withother compositions and/or procedures for the treatment of the conditionsdescribed above. For example, a tumor may be treated conventionally withsurgery, radiation, or chemotherapy combined with one or morecompositions of the present invention and then one or more compositionsof the present invention may be subsequently administered to the patientto extend the dormancy of micrometastases and to stabilize, inhibit, orreduce the growth of any residual primary tumor.

Compositions including TCN, TCN-P, TCN-PM and/or related compounds andone or more anthracycline analogs may be used with a sustained-releasematrix, which can be made of materials, usually polymers, which aredegradable by enzymatic or acid-based hydrolysis or by dissolution. Onceinserted into the body, the matrix is acted upon by enzymes and bodyfluids. A sustained-release matrix for example is chosen frombiocompatible materials such as liposomes, polylactides (polylacticacid), polyglycolide (polymer of glycolic acid), polylactideco-glycolide (copolymers of lactic acid and glycolic acid),polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid,collagen, chondroitin sulfate, carboxylic acids, fatty acids,phospholipids, polysaccharides, nucleic acids, polyamino acids, aminoacids such as phenylalanine, tyrosine, isoleucine, polynucleotides,polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferredbiodegradable matrix is a matrix of one of either polylactide,polyglycolide, or polylactide co-glycolide (co-polymers of lactic acidand glycolic acid).

The TCN, TCN-P, TCN-PM and/or related compounds and one or moreanthracycline analogs may also be administered in the form of liposomes.As is known in the art, liposomes are generally derived fromphospholipids or other lipid substances. Liposomes are formed by mono-or multi-lamellar hydrated liquid crystals that are dispersed in anaqueous medium. Any non-toxic, physiologically-acceptable andmetabolizable lipid capable of forming liposomes can be used. Theliposome can contain, in addition to one or more compositions of thepresent invention, stabilizers, preservatives, excipients, and the like.Examples of lipids are the phospholipids and the phosphatidyl cholines(lecithins), both natural and synthetic. Methods to form liposomes areknown in the art.

The TCN, TCN-P, TCN-PM and/or related compounds and one or moreanthracycline analogs may be formulated as aerosols for application,such as by inhalation. These formulations for administration to therespiratory tract can be in the form of an aerosol or solution for anebulizer, or as a microfine powder for insufflation, alone or incombination with an inert carrier such as lactose. In such a case, theparticles of the formulation will, in one embodiment, have diameters ofless than 50 microns, in one embodiment less than 10 microns.

Compositions including the TCN, TCN-P, TCN-PM and/or related compoundsand one or more anthracycline analogs may be used in combination withother compositions and/or procedures for the treatment of the conditionsdescribed above. For example. a tumor may be treated conventionally withsurgery, radiation, or chemotherapy combined with one or morecompositions of the present invention and then one or more compositionsof the present invention may be subsequently administered to the patientto extend the dormancy of micrometastases and to stabilize, inhibit, orreduce the growth of any residual primary tumor.

Compositions including TCN, TCN-P, TCN-PM and/or related compounds andan erlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof may be used with a sustained-release matrix, which can be madeof materials, usually polymers, which are degradable by enzymatic oracid-based hydrolysis or by dissolution. Once inserted into the body,the matrix is acted upon by enzymes and body fluids. A sustained-releasematrix for example is chosen from biocompatible materials such asliposomes, polylactides (polylactic acid), polyglycolide (polymer ofglycolic acid), polylactide co-glycolide (copolymers of lactic acid andglycolic acid), polyanhydrides, poly(ortho)esters, polypeptides,hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fattyacids, phospholipids, polysaccharides, nucleic acids, polyamino acids,amino acids such as phenylalanine, tyrosine, isoleucine,polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.A preferred biodegradable matrix is a matrix of one of eitherpolylactide, polyglycolide, or polylactide co-glycolide (co-polymers oflactic acid and glycolic acid).

The TCN, TCN-P, TCN-PM and/or related compounds and an erlotinib-likecompound, for example, gefitinib, erlotinib or a salt thereof may alsobe administered in the form of liposomes. As is known in the art,liposomes are generally derived from phospholipids or other lipidsubstances. Liposomes are formed by mono- or multi-lamellar hydratedliquid crystals that are dispersed in an aqueous medium. Any non-toxic,physiologically-acceptable and metabolizable lipid capable of formingliposomes can be used. The liposome can contain, in addition to one ormore compositions of the present invention, stabilizers, preservatives,excipients, and the like. Examples of lipids are the phospholipids andthe phosphatidyl cholines (lecithins), both natural and synthetic.Methods to form liposomes are known in the art.

The TCN, TCN-P, TCN-PM and/or related compounds and an erlotinib-likecompound, for example, gefitinib, erlotinib or a salt thereof may beformulated as aerosols for application, such as by inhalation. Theseformulations for administration to the respiratory tract can be in theform of an aerosol or solution for a nebulizer, or as a microfine powderfor insufflation, alone or in combination with an inert carrier such aslactose. In such a case, the particles of the formulation will, in oneembodiment, have diameters of less than 50 microns, in one embodimentless than 10 microns.

Compositions including the TCN, TCN-P, TCN-PM and/or related compoundsand an erlotinib-like compound, for example, gefitinib, erlotinib or asalt thereof may be used in combination with other compositions and/orprocedures for the treatment of the conditions described above. Forexample, a tumor may be treated conventionally with surgery, radiation,or chemotherapy combined with one or more compositions of the presentinvention and then one or more compositions of the present invention maybe subsequently administered to the patient to extend the dormancy ofmicrometastases and to stabilize, inhibit, or reduce the growth of anyresidual primary tumor.

Compositions including TCN, TCN-P, TCN-PM and/or related compounds andone or more platinum compounds may be used with a sustained-releasematrix, which can be made of materials, usually polymers, which aredegradable by enzymatic or acid-based hydrolysis or by dissolution. Onceinserted into the body, the matrix is acted upon by enzymes and bodyfluids. A sustained-release matrix for example is chosen frombiocompatible materials such as liposomes, polylactides (polylacticacid), polyglycolide (polymer of glycolic acid), polylactideco-glycolide (copolymers of lactic acid and glycolic acid),polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid,collagen, chondroitin sulfate, carboxylic acids, fatty acids,phospholipids, polysaccharides, nucleic acids, polyamino acids, aminoacids such as phenylalanine, tyrosine, isoleucine, polynucleotides,polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferredbiodegradable matrix is a matrix of one of either polylactide,polyglycolide, or polylactide co-glycolide (co-polymers of lactic acidand glycolic acid).

The TCN, TCN-P, TCN-PM and/or related compounds and one or more platinumcompounds may also be administered in the form of liposomes. As is knownin the art, liposomes are generally derived from phospholipids or otherlipid substances. Liposomes are formed by mono- or multi-lamellarhydrated liquid crystals that are dispersed in an aqueous medium. Anynon-toxic, physiologically-acceptable and metabolizable lipid capable offorming liposomes can be used. The liposome can contain, in addition toone or more compositions of the present invention, stabilizers,preservatives, excipients, and the like. Examples of lipids are thephospholipids and the phosphatidyl cholines (lecithins), both naturaland synthetic. Methods to form liposomes are known in the art.

The TCN, TCN-P, TCN-PM and/or related compounds and one or more platinumcompounds may be formulated as aerosols for application, such as byinhalation. These formulations for administration to the respiratorytract can be in the form of an aerosol or solution for a nebulizer, oras a microfine powder for insufflation, alone or in combination with aninert carrier such as lactose. In such a case, the particles of theformulation will, in one embodiment, have diameters of less than 50microns, in one embodiment less than 10 microns.

Compositions including the TCN, TCN-P, TCN-PM and/or related compoundsand one or more platinum compounds may be used in combination with othercompositions and/or procedures for the treatment of the conditionsdescribed above. For example, a tumor may be treated conventionally withsurgery, radiation, or chemotherapy combined with one or morecompositions of the present invention and then one or more compositionsof the present invention may be subsequently administered to the patientto extend the dormancy of micrometastases and to stabilize, inhibit, orreduce the growth of any residual primary tumor.

Compositions including TCN, TCN-P, TCN-PM and/or related compounds andbortezomib and derivatives thereof analogs may be used with asustained-release matrix, which can be made of materials, usuallypolymers, which are degradable by enzymatic or acid-based hydrolysis orby dissolution. Once inserted into the body, the matrix is acted upon byenzymes and body fluids. A sustained-release matrix for example ischosen from biocompatible materials such as liposomes, polylactides(polylactic acid), polyglycolide (polymer of glycolic acid), polylactideco-glycolide (copolymers of lactic acid and glycolic acid),polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid,collagen, chondroitin sulfate, carboxylic acids, fatty acids,phospholipids, polysaccharides, nucleic acids, polyamino acids, aminoacids such as phenylalanine, tyrosine, isoleucine, polynucleotides,polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferredbiodegradable matrix is a matrix of one of either polylactide,polyglycolide, or polylactide co-glycolide (co-polymers of lactic acidand glycolic acid).

The TCN, TCN-P, TCN-PM and/or related compounds and bortezomib andderivatives thereof analogs may also be administered in the form ofliposomes. As is known in the art, liposomes are generally derived fromphospholipids or other lipid substances. Liposomes are formed by mono-or multi-lamellar hydrated liquid crystals that are dispersed in anaqueous medium. Any non-toxic, physiologically-acceptable andmetabolizable lipid capable of forming liposomes can be used. Theliposome can contain, in addition to compositions of the presentinvention, stabilizers, preservatives, excipients, and the like.Examples of lipids are the phospholipids and the phosphatidyl cholines(lecithins), both natural and synthetic. Methods to form liposomes areknown in the art.

The TCN, TCN-P, TCN-PM and/or related compounds and bortezomib andderivatives thereof analogs may be formulated as aerosols forapplication, such as by inhalation. These formulations foradministration to the respiratory tract can be in the form of an aerosolor solution for a nebulizer, or as a microfine powder for insufflation,alone or in combination with an inert carrier such as lactose. In such acase, the particles of the formulation will, in one embodiment, havediameters of less than 50 microns, in one embodiment less than 10microns.

Compositions including the TCN, TCN-P, TCN-PM and/or related compoundsand bortezomib and derivatives thereof analogs may be used incombination with other compositions and/or procedures for the treatmentof the conditions described above. For example, a tumor may be treatedconventionally with surgery, radiation, or chemotherapy combined withcompositions of the present invention and then compositions of thepresent invention may be subsequently administered to the patient toextend the dormancy of micrometastases and to stabilize, inhibit, orreduce the growth of any residual primary tumor.

7.1. Additional Embodiments

The pharmaceutical compositions including TCN, TCN-P, TCN-PM and/orrelated compounds and one or more taxanes can be formulated according toknown methods for preparing pharmaceutically useful compositions.Formulations are described in a number of sources which are well knownand readily available to those skilled in the art. For example,Remington's Pharmaceutical Sciences (Martin E W [1995] Easton Pa., MackPublishing Company, 19^(th) ed.) describes formulations which can beused in connection with the subject invention. Formulations suitable foradministration include, for example, aqueous sterile injectionsolutions, which may contain antioxidants, buffers, bacteriostats, andsolutes which render the formulation isotonic with the blood of theintended recipient; and aqueous and nonaqueous sterile suspensions whichmay include suspending agents and thickening agents. The formulationsmay be presented in unit-dose or multi-dose containers, for examplesealed ampoules and vials, and may be stored in a freeze dried(lyophilized) condition requiring only the condition of the sterileliquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, theformulations of the subject invention can include other agentsconventional in the art having regard to the type of formulation inquestion.

The pharmaceutical compositions including TCN, TCN-P, TCN-PM and/orrelated compounds and a molecule that modulates the HER2/neu (erbB2)receptor, for example, trastuzumab or a salt thereof, can be formulatedaccording to known methods for preparing pharmaceutically usefulcompositions. Formulations are described in a number of sources whichare well known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa. describes formulations which can be used in connection withthe subject invention. Formulations suitable for administration include,for example, aqueous sterile injection solutions, which may containantioxidants, buffers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and nonaqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze dried (lyophilized) conditionrequiring only the condition of the sterile liquid carrier, for example,water for injections, prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powder, granules, tablets,etc. It should be understood that in addition to the ingredientsparticularly mentioned above, the formulations of the subject inventioncan include other agents conventional in the art having regard to thetype of formulation in question.

The pharmaceutical compositions including TCN, TCN-P, TCN-PM and/orrelated compounds and one or more anthracycline analogs can beformulated according. to known methods for preparing pharmaceuticallyuseful compositions. Formulations are described in a number of sourceswhich are well known and readily available to those skilled in the art.For example, Remington's Pharmaceutical Sciences (Martin E W [1995]Easton Pa., Mack Publishing Company, 19^(th) ed.) describes formulationswhich can be used in connection with the subject invention. Formulationssuitable for administration include, for example, aqueous sterileinjection solutions, which may contain antioxidants, buffers,bacteriostats, and solutes which render the formulation isotonic withthe blood of the intended recipient; and aqueous and nonaqueous sterilesuspensions which may include suspending agents and thickening agents.The formulations may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in a freezedried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, theformulations of the subject invention can include other agentsconventional in the art having regard to the type of formulation inquestion.

The pharmaceutical compositions including TCN, TCN-P, TCN-PM and/orrelated compounds and an erlotinib-like compound, for example,gefitinib, erlotinib or a salt thereof can be formulated according toknown methods for preparing pharmaceutically useful compositions.Formulations are described in a number of sources which are well knownand readily available to those skilled in the art. For example,Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa. describes formulations which can be used in connection with thesubject invention. Formulations suitable for administration include, forexample, aqueous sterile injection solutions, which may containantioxidants, buffers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and nonaqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze dried (lyophilized) conditionrequiring only the condition of the sterile liquid carrier, for example,water for injections, prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powder, granules, tablets,etc. It should be understood that in addition to the ingredientsparticularly mentioned above, the formulations of the subject inventioncan include other agents conventional in the art having regard to thetype of formulation in question.

The pharmaceutical compositions including TCN, TCN-P, TCN-PM and/orrelated compounds and one or more platinum compounds can be formulatedaccording to known methods for preparing pharmaceutically usefulcompositions. Formulations are described in a number of sources whichare well known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Sciences (Mack Publishing Company,Easton. Pa.) describes formulations which can be used in connection withthe subject invention. Formulations suitable for administration include,for example, aqueous sterile injection solutions, which may containantioxidants, buffers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and nonaqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze dried (lyophilized) conditionrequiring only the condition of the sterile liquid carrier, for example,water for injections, prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powder, granules, tablets,etc. It should be understood that in addition to the ingredientsparticularly mentioned above, the formulations of the subject inventioncan include other agents conventional in the art having regard to thetype of formulation in question.

The pharmaceutical compositions including TCN. TCN-P, TCN-PM and/orrelated compounds and bortezomib and derivatives thereof analogs can beformulated according to known methods for preparing pharmaceuticallyuseful compositions. Formulations are described in a number of sourceswhich are well known and readily available to those skilled in the art.For example, Remington's Pharmaceutical Sciences (Martin E W [1995]Easton Pa., Mack Publishing Company, 19^(th) ed.) describes formulationswhich can be used in connection with the subject invention. Formulationssuitable for administration include, for example, aqueous sterileinjection solutions, which may contain antioxidants, buffers,bacteriostats, and solutes which render the formulation isotonic withthe blood of the intended recipient; and aqueous and nonaqueous sterilesuspensions which may include suspending agents and thickening agents.The formulations may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in a freezedried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, theformulations of the subject invention can include other agentsconventional in the art having regard to the type of formulation inquestion.

The methods of the present invention, for example, for inhibiting thegrowth of a cancerous cell, can be advantageously combined with at leastone additional therapeutic method, including but not limited tochemotherapy, radiation therapy, therapy that selectively inhibits Rasoncogenic signaling, or any other therapy known to those of skill in theart of the treatment and management of cancer, such as administration ofan anti-cancer agent.

Administration of TCN, TCN-P, TCN-PM and/or related compounds as a saltmay be carried out. Examples of pharmaceutically acceptable salts areorganic acid addition salts formed with acids which form a physiologicalacceptable anion, for example, tosylate, methanesulfonate, acetate,citrate, malonate, tartarate, succinate, benzoate, ascorbate,alpha-ketoglutarate, and alpha-glycerophosphate. Suitable inorganicsalts may also be formed, including hydrochloride, sulfate, nitrate,bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

The TCN, TCN-P, TCN-PM and/or related compounds and one or more taxanescan be formulated as pharmaceutical compositions and administered to asubject, such as a human or veterinary patient, in a variety of formsadapted to the chosen route of administration, i.e., orally orparenterally, by intravenous, intramuscular, topical or subcutaneousroutes.

Thus, the TCN, TCN-P, TCN-PM and/or related compounds and one or moretaxanes of the present invention may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle (i.e.,carrier) such as an inert diluent or an assimilable edible carrier. Theymay be enclosed in hard or soft shell gelatin capsules, may becompressed into tablets, or may be incorporated directly with the foodof the patient's diet. For oral therapeutic administration, thecompounds may be combined with one or more excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active agent. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of the active compound insuch therapeutically useful compositions is such that an effectivedosage level will be obtained.

The TCN, TCN-P, TCN-PM and/or related compounds and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, can be formulated as pharmaceutical compositions andadministered to a subject, such as a human or veterinary patient, in avariety of forms adapted to the chosen route of administration, i.e.,orally or parenterally, by intravenous, intramuscular, topical orsubcutaneous routes.

Thus, the TCN, TCN-P, TCN-PM and/or related compounds and a moleculethat modulates the HER2/neu (erbB2) receptor, for example, trastuzumabor a salt thereof, of the present invention may be systemicallyadministered, e.g., orally, in combination with a pharmaceuticallyacceptable vehicle (i.e., carrier) such as an inert diluent or anassimilable edible carrier. They may be enclosed in hard or soft shellgelatin capsules, may be compressed into tablets, or may be incorporateddirectly with the food of the patient's diet. For oral therapeuticadministration, the compounds may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% ofactive agent. The percentage of the compositions and preparations may,of course, be varied and may conveniently be between about 2 to about60% of the weight of a given unit dosage form. The amount of the activecompound in such therapeutically useful compositions is such that aneffective dosage level will be obtained.

The TCN, TCN-P, TCN-PM and/or related compounds and one or moreanthracycline analogs can be formulated as pharmaceutical compositionsand administered to a subject, such as a human or veterinary patient, ina variety of forms adapted to the chosen route of administration, i.e.,orally or parenterally, by intravenous, intramuscular, topical orsubcutaneous routes.

Thus, the TCN, TCN-P, TCN-PM and/or related compounds and one or moreanthracycline analogs of the present invention may be systemicallyadministered, e.g., orally, in combination with a pharmaceuticallyacceptable vehicle (i.e., carrier) such as an inert diluent or anassimilable edible carrier. They may be enclosed in hard or soft shellgelatin capsules, may be compressed into tablets, or may be incorporateddirectly with the food of the patient's diet. For oral therapeuticadministration, the compounds may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% ofactive agent. The percentage of the compositions and preparations may,of course, be varied and may conveniently be between about 2 to about60% of the weight of a given unit dosage form. The amount of the activecompound in such therapeutically useful compositions is such that aneffective dosage level will be obtained.

The TCN, TCN-P, TCN-PM and/or related compounds and an erlotinib-likecompound, for example, gefitinib, erlotinib or a salt thereof can beformulated as pharmaceutical compositions and administered to a subject,such as a human or veterinary patient, in a variety of forms adapted tothe chosen route of administration, i.e., orally or parenterally, byintravenous, intramuscular, topical or subcutaneous routes.

Thus, the TCN, TCN-P, TCN-PM and/or related compounds and anerlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof of the present invention may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle (i.e.,carrier) such as an inert diluent or an assimilable edible carrier. Theymay be enclosed in hard or soft shell gelatin capsules, may becompressed into tablets, or may be incorporated directly with the foodof the patient's diet. For oral therapeutic administration, thecompounds may be combined with one or more excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active agent. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of the active compound insuch therapeutically useful compositions is such that an effectivedosage level will be obtained.

The TCN, TCN-P, TCN-PM and/or related compounds and one or more platinumcompounds can be formulated as pharmaceutical compositions andadministered to a subject, such as a human or veterinary patient, in avariety of forms adapted to the chosen route of administration, i.e.,orally or parenterally, by intravenous, intramuscular, topical orsubcutaneous routes.

Thus, the TCN, TCN-P, TCN-PM and/or related compounds and one or moreplatinum compounds of the present invention may be systemicallyadministered, e.g., orally, in combination with a pharmaceuticallyacceptable vehicle (i.e., carrier) such as an inert diluent or anassimilable edible carrier. They may be enclosed in hard or soft shellgelatin capsules, may be compressed into tablets, or may be incorporateddirectly with the food of the patient's diet. For oral therapeuticadministration, the compounds may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% ofactive agent. The percentage of the compositions and preparations may,of course, be varied and may conveniently be between about 2 to about60% of the weight of a given unit dosage form. The amount of the activecompound in such therapeutically useful compositions is such that aneffective dosage level will be obtained.

The TCN, TCN-P, TCN-PM and/or related compounds and bortezomib andderivatives thereof analogs can be formulated as pharmaceuticalcompositions and administered to a subject, such as a human orveterinary patient, in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, the TCN, TCN-P, TCN-PM and/or related compounds and bortezomib andderivatives thereof analogs of the present invention may be systemicallyadministered, e.g., orally, in combination with a pharmaceuticallyacceptable vehicle (i.e., carrier) such as an inert diluent or anassimilable edible carrier. They may be enclosed in hard or soft shellgelatin capsules, may be compressed into tablets, or may be incorporateddirectly with the food of the patient's diet. For oral therapeuticadministration, the compounds may be combined with excipients and usedin the form of ingestible tablets, buccal tablets, troches, capsules,elixirs, suspensions, syrups, wafers, and the like. Such compositionsand preparations should contain at least 0.1% of active agent. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of the active compound insuch therapeutically useful compositions is such that an effectivedosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the compounds of the invention, sucrose or fructose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any unit dosage form should be pharmaceutically acceptableand substantially non-toxic in the amounts employed. In addition, thecompounds of the invention may be incorporated into sustained-releasepreparations and devices.

The TCN, TCN-P, TCN-PM and/or related compounds and one or more taxanesmay also be administered intravenously or intraperitoneally by infusionor injection. Solutions of the active agents or their salts can beprepared in water, optionally mixed with a nontoxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, triacetin, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The TCN, TCN-P, TCN-PM and/or related compounds and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activeagents or their salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The TCN, TCN-P, TCN-PM and/or related compounds and one or moreanthracycline analogs may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activeagents or their salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The TCN, TCN-P, TCN-PM and/or related compounds and an erlotinib-likecompound, for example, gefitinib, erlotinib or a salt thereof may alsobe administered intravenously or intraperitoneally by infusion orinjection. Solutions of the active agents or their salts can be preparedin water, optionally mixed with a nontoxic surfactant. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, triacetin,and mixtures thereof and in oils. Under ordinary conditions of storageand use, these preparations contain a preservative to prevent the growthof microorganisms.

The TCN, TCN-P, TCN-PM and/or related compounds and one or more platinumcompounds may also be administered intravenously or intraperitoneally byinfusion or injection. Solutions of the active agents or their salts canbe prepared in water, optionally mixed with a nontoxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, triacetin, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The TCN, TCN-P, TCN-PM and/or related compounds and bortezomib andderivatives thereof analogs may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activeagents or their salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powdersincluding the active ingredient which are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. In all cases, the ultimate dosageform must be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium including, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, buffers or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating compounds ofthe invention in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

For topical administration, the TCN, TCN-P, TCN-PM and/or relatedcompounds and one or more taxanes may be applied in pure-form, i.e.,when they are liquids. However, it will generally be desirable toadminister them to the skin as compositions or formulations, incombination with a dermatologically acceptable carrier, which may be asolid or a liquid.

For topical administration, the TCN, TCN-P, TCN-PM and/or relatedcompounds and a molecule that modulates the HER2/neu (erbB2) receptor,for example, trastuzumab or a salt thereof, may be applied in pure-form,i.e., when they are liquids. However, it will generally be desirable toadminister them to the skin as compositions or formulations, incombination with a dermatologically acceptable carrier, which may be asolid or a liquid.

For topical administration, the TCN, TCN-P, TCN-PM and/or relatedcompounds and one or more anthracycline analogs may be applied inpure-form, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

For topical administration, the TCN, TCN-P, TCN-PM and/or relatedcompounds and an erlotinib-like compound, for example, gefitinib,erlotinib or a salt thereof may be applied in pure-form, i.e., when theyare liquids. However, it will generally be desirable to administer themto the skin as compositions or formulations, in combination with adermatologically acceptable carrier, which may be a solid or a liquid.

For topical administration, the TCN, TCN-P, TCN-PM and/or relatedcompounds and one or more platinum compounds may be applied inpure-form, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

For topical administration, the TCN, TCN-P, TCN-PM and/or relatedcompounds and bortezomib and derivatives thereof analogs may be appliedin pure-form, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the compounds of the invention can be dissolved ordispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants such as fragrances and additional antimicrobialagents can be added to optimize the properties for a given use. Theresultant liquid compositions can be applied from absorbent pads, usedto impregnate bandages and other dressings, or sprayed onto the affectedarea using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user. Examples of useful dermatological compositionswhich can be used to deliver the compounds of the invention to the skinare disclosed in Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S.Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Woltzman(U.S. Pat. No. 4,820,508).

Useful dosages of the pharmaceutical compositions of the presentinvention can be determined by comparing their in vitro activity, and inviva activity in animal models. Methods for the extrapolation ofeffective dosages in mice, and other animals, to humans are known to theart; for example, see U.S. Pat. No. 4,938,949.

In one non-limiting embodiment, the concentration of the active agent ina liquid composition, such as a lotion, can be from about 0.1-25 wt-%,or from about 0.5-10 wt.-%. In one embodiment, the concentration in asemi-solid or solid composition such as a gel or a powder can be about0.1-5 wt.-%, preferably about 0.5-2.5 wt.-%. In one embodiment, singledosages for injection, infusion or ingestion will generally vary between5-1500 mg, and may be administered, i.e., 1-3 times daily, to yieldlevels of about 0.1-50 mg/kg, for adults. A non-limiting dosage of thepresent invention can be between 7.5 to 45 mg per clay, administeredorally, with appropriate adjustment for the body weight of anindividual.

Accordingly, the invention includes a pharmaceutical compositionincluding TCN, TCN-P, TCN-PM and/or related compounds and one or moretaxanes or pharmaceutically acceptable salts thereof, in combinationwith a pharmaceutically acceptable carrier. Pharmaceutical compositionsadapted for oral, topical or parenteral administration, including anamount of TCN, TCN-P, TCN-PM and/or related compounds and one or moretaxanes or a pharmaceutically acceptable salt thereof, constitute apreferred embodiment of the invention. The dose administered to asubject, particularly a human, in the context of the present inventionshould be sufficient to affect a therapeutic response in the patientover a reasonable time frame. One skilled in the art will recognize thatdosage will depend upon a variety of factors including the condition ofthe animal, the body weight of the animal, as well as the severity andstage of the cancer.

A suitable dose is that which will result in a concentration of the TCN,TCN-P, TCN-PM and/or related compounds and one or more taxanes in tumortissue which is known to affect the desired response. The preferreddosage is the amount which results in maximum inhibition of cancer cellgrowth, without unmanageable side effects. Administration of TCN, TCN-P,TCN-PM and/or related compounds (or a pharmaceutically acceptable saltthereof) can be continuous or at distinct intervals, as can bedetermined by a person of ordinary skill in the art.

The invention also includes a pharmaceutical composition including TCN,TCN-P, TCN-PM and/or related compounds and a molecule that modulates theHER2/neu (erbB2) receptor, for example, trastuzumab or a salt thereof,or pharmaceutically acceptable salts thereof, in combination with apharmaceutically acceptable carrier. Pharmaceutical compositions adaptedfor oral, topical or parenteral administration, including an amount ofTCN, TCN-P, TCN-PM and/or related compounds and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, or a pharmaceutically acceptable salt thereof, constitutea preferred embodiment of the invention. The dose administered to asubject, particularly a human, in the context of the present inventionshould be sufficient to affect a therapeutic response in the patientover a reasonable time frame. One skilled in the art will recognize thatdosage will depend upon a variety of factors including the condition ofthe animal, the body weight of the animal, as well as the severity andstage of the cancer.

A suitable dose is that which will result in a concentration of the TCN,TCN-P, TCN-PM and/or related compounds and a molecule that modulates theHER2/neu (erbB2) receptor, for example, trastuzumab, in tumor tissuewhich is known to affect the desired response. The preferred dosage isthe amount which results in maximum inhibition of cancer cell growth,without unmanageable side effects. Administration of API-2 (or apharmaceutically acceptable salt thereof) can be continuous or atdistinct intervals, as can be determined by a person of ordinary skillin the art.

The invention also includes a pharmaceutical composition including TCN,TCN-P, TCN-PM and/or related compounds and one or more anthracyclineanalogs or pharmaceutically acceptable salts thereof, in combinationwith a pharmaceutically acceptable carrier. Pharmaceutical compositionsadapted for oral, topical or parenteral administration, including anamount of TCN, TCN-P, TCN-PM and/or related compounds and one or moreanthracycline analogs or a pharmaceutically acceptable salt thereof,constitute a preferred embodiment of the invention. The doseadministered to a subject, particularly a human, in the context of thepresent invention should be sufficient to affect a therapeutic responsein the patient over a reasonable time frame. One skilled in the art willrecognize that dosage will depend upon a variety of factors includingthe condition of the animal, the body weight of the animal, as well asthe severity and stage of the cancer.

A suitable dose is that which will result in a concentration of the TCN,TCN-P, TCN-PM and/or related compounds and one or more anthracyclineanalogs in tumor tissue which is known to affect the desired response.The preferred dosage is the amount which results in maximum inhibitionof cancer cell growth, without unmanageable side effects. Administrationof API-2 (or a pharmaceutically acceptable salt thereof) can becontinuous or at distinct intervals, as can be determined by a person ofordinary skill in the art.

The invention also includes a pharmaceutical composition including TCN,TCN-P, TCN-PM and/or related compounds and an erlotinib-like compound,for example, gefitinib, erlotinib or a salt thereof in combination witha pharmaceutically acceptable carrier. Pharmaceutical compositionsadapted for oral, topical or parenteral administration, including anamount of TCN, TCN-P, TCN-PM and/or related compounds and anerlotinib-like compound, for example, gefitinib, erlotinib or a saltthereof, constitute a preferred embodiment of the invention. The doseadministered to a subject, particularly a human, in the context of thepresent invention should be sufficient to affect a therapeutic responsein the patient over a reasonable time frame. One skilled in the art willrecognize that dosage will depend upon a variety of factors includingthe condition of the animal, the body weight of the animal, as well asthe severity and stage of the cancer.

A suitable dose is that which will result in a concentration of the TCN,TCN-P, TCN-PM and/or related compounds and an erlotinib-like compound,for example. gefitinib, erlotinib or a salt thereof in tumor tissuewhich is known to affect the desired response. The preferred dosage isthe amount which results in maximum inhibition of cancer cell growth,without unmanageable side effects. Administration of API-2 (or apharmaceutically acceptable salt thereof) can be continuous or atdistinct intervals, as can be determined by a person of ordinary skillin the art.

The invention also includes a pharmaceutical composition including TCN,TCN-P, TCN-PM and/or related compounds and one or more platinumcompounds or pharmaceutically acceptable salts thereof, in combinationwith a pharmaceutically acceptable carrier. Pharmaceutical compositionsadapted for oral, topical or parenteral administration, including anamount of TCN, TCN-P, TCN-PM and/or related compounds and one or moreplatinum compounds or a pharmaceutically acceptable salt thereof,constitute a preferred embodiment of the invention. The doseadministered to a subject, particularly a human, in the context of thepresent invention should be sufficient to affect a therapeutic responsein the patient over a reasonable time frame. One skilled in the art willrecognize that dosage will depend upon a variety of factors includingthe condition of the animal, the body weight of the animal, as well asthe severity and stage of the cancer.

A suitable dose is that which will result in a concentration of the TCN,TCN-P, TCN-PM and/or related compounds and one or more platinumcompounds in tumor tissue which is known to affect the desired response.The preferred dosage is the amount which results in maximum inhibitionof cancer cell growth, without unmanageable side effects. Administrationof API-2 (or a pharmaceutically acceptable salt thereof) can becontinuous or at distinct intervals, as can be determined by a person ofordinary skill in the art.

The invention also includes a pharmaceutical composition including TCN,TCN-P, TCN-PM and/or related compounds and bortezomib and derivativesthereof analogs or pharmaceutically acceptable salts thereof, incombination with a pharmaceutically acceptable carrier. Pharmaceuticalcompositions adapted for oral, topical or parenteral administration,including an amount of TCN, TCN-P, TCN-PM and/or related compounds andbortezomib and derivatives thereof analogs or a pharmaceuticallyacceptable salt thereof, constitute a preferred embodiment of theinvention. The dose administered to a subject, particularly a human, inthe context of the present invention should be sufficient to affect atherapeutic response in the patient over a reasonable time frame. Oneskilled in the art will recognize that dosage will depend upon a varietyof factors including the condition of the animal, the body weight of theanimal, as well as the severity and stage of the cancer.

A suitable dose is that which will result in a concentration of the TCN,TCN-P, TCN-PM and/or related compounds and bortezomib and derivativesthereof analogs in tumor tissue which is known to affect the desiredresponse. The preferred dosage is the amount which results in maximuminhibition of cancer cell growth, without unmanageable side effects.Administration of API-2 (or a pharmaceutically acceptable salt thereof)can be continuous or at distinct intervals, as can be determined by aperson of ordinary skill in the art.

Mammalian species which benefit from the disclosed methods for theinhibition of cancer cell growth, include, but are not limited to,primates, such as apes, chimpanzees, orangutans, humans, monkeys;domesticated animals (e.g., pets) such as dogs, cats, guinea pigs,hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets;domesticated farm animals such as cows, buffalo, bison, horses, donkey,swine, sheep, and goats; exotic animals typically found in zoos, such asbear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros,giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs,koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals, sealions, elephant seals, otters, porpoises, dolphins, and whales. Theterms “patient” and “subject” are used herein interchangeably and areintended to include such human and non-human mammalian species.Likewise, in vitro methods of the present invention can be earned out oncells of such mammalian species.

Patients in need of treatment using the methods of the present inventioncan be identified using standard techniques known to those in themedical profession.

The following examples are offered by way of illustration and not by wayof limitation.

8. EXAMPLES 8.1. Example 1 In Vitro Screening

Cell Lines and NCI Diversity Set. All cell lines can be purchased fromATCC or described previously (Cheng, J. Q., et al. Oncogene, 14:2793-2801, 1997, West, K. A., et al. Drug Resist. Updat., 5: 234-248,2002, Satyamoorthy, K., et al. Cancer Res. 61: 7318-7324, 2001). The NCIStructural Diversity Set is a library of 1,992 compounds selected fromthe approximately 140,000-compound NCI drug depository. In-depth data onthe selection, structures, and activities of these diversity setcompounds can be found on the NCI Developmental Therapeutics Program website.

Screening for Inhibition of Akt-transformed Cell Growth. AKT2transformed NIH3T3 cells or LXSN vector-transfected NIH3T3 control cells(Cheng, J. Q., et al. Oncogene, 14: 2793-2801, 1997) are plated into96-well tissue culture plate. Following treatment with 5 μM of NCIDiversity Set compound, cell growth can be detected with CellTier 96 OneSolution Cell Proliferation kit (Promega). Compounds that inhibit growthin AKT2-transformed but not LXSN-transfected NIH3T3 cells are consideredas candidates of Akt inhibitor and subjected to further analysis.

In vitro Protein Kinase, Cell Survival and Apoptosis Assays. In vitrokinase is performed as previously described (see, for example, Jiang,K., Coppola, ct al. Mol. Cell. Biol., 20:139-148, 2000). Cell survivalis assayed with MTS (Promega). Apoptosis was detected with annexin V,which is performed as previously described (Jiang, K., Coppola, et al.Mol. Cell. Biol., 20:139-148, 2000). Recombinant Akt and PDKI arepurchased from Upstate Biotechnology Inc.

Results

Identification of Small Molecule Inhibitor of Akt Signaling Pathway,API-2. Frequent alterations of Akt has been detected in human cancer anddisruption of Akt pathway induces apoptosis and inhibits rumor growth(Jetzt, A., et al. Cancer Res., 63: 697-706, 2003). Thus, Aki isconsidered as an attractive molecular target for development of novelcancer therapeutics. To identify small molecule inhibitor(s) of Akt, achemical library of 1,992-compounds from the NCI (the NCI Diversity Set)is evaluated for agents capable of inhibition of growth inAKT2-transformed but not empty vector LXSN-transfected NIH3T3 cells.Repeated experiments showed that 32 compounds inhibited growth only inAKT2-transformed cells. The most potent of these compounds, API-2 (NCIidentifier: NSI 154020), can suppress cell growth at a concentration of50 nM. FIG. 1A shows the chemical structure of API-2, which is alsoknown as triciribine (Schweinsberg, P. D., et al. Biochem Pharmacol.,30: 2521-2526, 1981). The fact that API-2 inhibits selectively AKT-2transformed cells over untransformed parental cells prompted us todetermine whether API-2 is an inhibitor of AKT2 kinase. To this end,AKT2 is immunoprecipitated with anti-AKT2 antibody from AKT-2transformed NIH3T3 cells following treatment with API-2. AKT2immunoprecipitates were immunoblotted with anti-phospho-Akt antibodies.As shown in FIG. 1B, API-2 significantly inhibited AKT2 phosphorylationat both threonine-309 and serine-474, which are required for fullactivation of AKT2 (Datta, S. R., et al. Genes Dev. 13: 2905-2927.1999). As three isoforms of Akt share high homology and similarstructure, the effect of API-2 on their kinase activities is evaluated.HEK293 cells are transfected with HA-Akt 1, -AKT2 and -AKT3,serum-starved overnight and treated with API-2 for 60 min prior to EGF(50 ng/ml) stimulation. Triple experiments showed that API-2 suppressedEGF-induced kinase activity and phosphorylation of Akt1, AKT2 and AKT3(FIG. 1C). However, kinase activity of recombinant constitutively activeAKT2 (Myr-AKT2) is not inhibited by API-2 in an in vitro kinase reaction(FIG. 1D), suggesting that API-2 does not directly inhibit Akt in vitroand that API-2 neither functions as ATP competitor nor as the substratecompetitor that binds to active site of Akt.

API-2 does not Inhibit Known Upstream Activators of Akt. It has beenwell documented that Akt is activated by extracellular stimuli andintracellular signal molecules, such as active Ras and Src, through aPI3K-dependent manner. Therefore, API-2 inhibition of Akt could resultfrom targeting upstream molecule(s) of Akt. As PI3K and PDKI are directupstream regulators of Akt (Datta, S. R., et al. Genes Dev. 13:2905-2927, 1999), whether API-2 inhibits PI3K and/or PDK1 is examined.HEK293 cells are serum-starved and then can be treated with API-2 orPI3K inhibitor, wortmannin, for 30 min prior to EGF stimulation. PI3K isimmunoprecipitated with anti-p110α antibody. The immunoprecipitates aresubjected to in vitro PI3K kinase assay using PI-4-P as a substrate. Asshown in FIG. 2A, the EGF-induced PI3K activity is inhibited bywortmannin but not by API-2. To evaluate the effect of API-2 on PDK1, anassay in which recombinant PDK1 promotes the threonine-309phosphorylation of AKT2 peptides is used in the presence of lipidvesicles containing phosphotidylinositol. As shown in FIG. 2B, the assayis potently inhibited by the control PDK1 inhibitor staurosporine(IC50=5 nM). In contrast, API-2 displayed only 21% inhibition of theassay at the highest concentration tested (5.1 μM). To further evaluatethe effect of API-2 on PDKI activation, the autophosphorylation level ofPDKI at serine-241, a residue that is phosphorylated by itself and iscritical for its activity is examined (Datta, S. R., et al. Genes Dev.13: 2905-2927, 1999), following API-2 treatment of HEK293 cells.Triplicate experiments show that phosphorylation levels of PDK1 are notinhibited by API-2 (FIG. 2B). However, PI3K inhibitor wortmannin caninhibit EGF-stimulated PDKI (FIG. 2B).

API-2 is Highly Selective for the Akt over PKC, PKA, SGK, STAT, JNK,p38, and ERK Signaling Pathways. Akt belongs to AGC(PKA/PKG/PKC) kinasefamily, which also include PKA, PKC, serum- and glucocorticoid-induciblekinase (SGK), p90 ribosomal S6 kinase, p70S6K, mitogen- andstress-activated protein kinase and PKC-related kinase. Among AGC kinasefamily, protein structures of PKA, PKC and SGK are more close to Aktkinase than other members. Therefore, next examined are the effects ofAPI-2 on the enzymatic activities of these 3 kinases. HEK293 cells aretransfected with HA-tagged PKA, PKCα or SGK. In vitro kinase assay andimmunoblotting analysis show that the kinase activities of PKA and PKCαare inhibited by PKA1 and Ro 31-8220, a PKC inhibitor, respectively,whereas API-2 exhibits no effect on their activities (FIGS. 2C and 2E).Further, serum-induced SGK kinase activity is attenuated by wortmanninbut not by API-2 (FIG. 2D). In addition, it is determined whether API-2has effect on other oncogenic survival pathways. Western blottinganalyses with commercially available anti-phospho-antibodies revealsthat phosphorylation levels of Stat3, JNK, p38 and Erk1/2 were notaffected by API-2 treatment (FIG. 2F). These data indicate that API-2specifically inhibits Akt signaling pathway.

API-2 Suppresses Cell Growth and Induces Apoptosis inAkt-overexpressing/activating Human Cancer Cell Lines. The ability ofAPI-2 to selectively inhibit the Akt pathway suggests that it shouldinhibit proliferation and/or induces apoptosis preferentially in thosetumor cells with aberrant expression/activation of Akt. As activation ofAkt in human malignancies commonly results from overexpression of Akt orPTEN mutations, API-2 is used to treat the cells that expressconstitutively active Akt, caused by overexpression of AKT2 (OVCAR3,OVCAR8, PANC1 and AKT2-transformed NIH3T3) or mutations of the PTEN gene(PC-3, LNCaP, MDA-MB-468), and cells that do not (OVCAR5, DU-145, T47D,COLO357 and LXSN-NIH3T3) as well as melanoma cells that are activated byIGF-1 to activate Akt or do not respond to growth stimulation by IGF-1(Satyamoorthy, K., et al. Cancer Res. 61: 7318-7324, 2001).Immunoblotting analysis showed that phosphorylation levels of Akt areinhibited by API-2 only in the cells expressing elevated Akt orresponding to IGF-1 simulation (FIG. 3A). Accordingly, API-2 inhibitedcell growth to a much higher degree in Akt-overexpressing/activatingcells as compared to those with low levels of Akt. As shown in FIG. 3B,API-2 treatment inhibited cell proliferation by approximate 50-60% inAkt-overexpressing/activating cell lines, LNCaP, PC-3, OVCAR3, OVCA8,PANC1, MDA-MB-468, and WM35, whereas only by about 10-20% in DU145,OVCAR5, COLO357, T47D and WM852 cells, which exhibit low levels of Aktor do not respond to growth stimulation by IGF-1. Moreover, API-2induces apoptosis by 8-fold (OVCAR3), 6-fold (OVCAR8), 6-fold (PANC1),and 3-fold (AKT2-NIH3T3). No significant difference of apoptosis isobserved between API-2 and vehicle (DMSO) treatment in OVCAR5, COLO357and LXSN-NIH3T3 cells. Thus, API-2 inhibits cell growth and inducesapoptosis preferentially in cells that express aberrant Akt.

API-2 in combination with Taxol Acts Synergistically to Suppress CellGrowth and Induce Apoptosis in Akt-overexpressing/activating HumanCancer Cell Lines. The ability of API-2 and Taxol to selectively inhibitthe Akt pathway suggests that it should inhibit proliferation and/orinduces apoptosis preferentially in those tumor cells with aberrantexpression/activation of Akt. As activation of Akt in human malignanciescommonly results from overexpression of Akt or PTEN mutations, API-2 andTaxol in combination are used to treat the cells that expressconstitutively active Akt, caused by overexpression of AKT2 (OVCAR3,OVCAR8, PANC1 and AKT2-transformed NIH3T3) or mutations of the PTEN gene(PC-3, LNCaP, MDA-MB-468), and cells that do not (OVCAR5, DU-145, T47D,COLO357 and LXSN-NIH3T3) as well as melanoma cells that are activated byIGF-1 to activate Akt or do not respond to growth stimulation by IGF-1.(FIG. 3A). API-2 and Taxol inhibits cell growth to a much higher degreein Akt-overexpressing/activating cells as compared to those with lowlevels of Akt. As shown in FIG. 9, API-2/Taxol treatment inhibits cellproliferation by approximate 50-65% in Akt-overexpressing/activatingcell lines, LNCaP, PC-3, OVCAR3, OVCA8, PANC1, MDA-MB-468, and WM35,whereas only by about 10-20% in DU145, OVCAR5, COLO357, T47D and WM852cells, which exhibit low levels of Akt or do not respond to growthstimulation by IGF-1. Thus, API-2 and Taxol acts synergistically toinhibit cell growth and induce apoptosis preferentially in cells thatexpress aberrant Akt.

API-2 Inhibits Downstream Targets of Akt. It has been shown that Aktexerts its cellular effects through phosphorylation of a number ofproteins (Datta, S. R., et al. Genes Dev. 13: 2905-2927, 1999). Morethan 20 proteins have been identified as Akt substrates, including themembers of Forkhead protein family (FKHR, AFX and FKHRL1),tuberlin/TSC2, p70^(S6K), GSK-3□, p21^(WAF1/Cip1), p27^(kip1), MDM2,Bad, ASK1 and IKKαetc. It is next examined whether API-2 inhibitsdownstream targets of Akt. As anti-phospho-tuberlin, -Bad, -AFX, and-GSK-3□ antibodies are commercially available, therefore, the effect ofAPI-2 on their phosphorylation induced by Akt was determined. FollowingAPI-2 (1 μM) treatment, OVCAR3 cells was lysed and immunoblotted withthe individual anti-phospho-antibody. FIG. 4A shows that API-2considerably inhibited the phosphorylation levels of tuberlin leading tostabilization and upregulation of tuberin (Dan, H. C., et al. J. Biol.Chem., 277: 35364-35370. 2002). The phosphorylation levels of Bad,(GSK-3 . . . , and AFX are partially attenuated by API-2. These datasuggest that API-2 induces cell death and cell growth arrest byinhibiting phosphorylation of its downstream targets. API-2 inhibitionof Akt downstream targets at different degrees could be due to the factthat phosphorylation sites of these targets are also regulated by otherkinase(s), for instance, Bad serine-136 is phosphorylated by PAK1 inaddition to Akt (Schurmann, A., et al. Mol. Cell. Biol., 20: 453-461,2000).

8.2 Example 2 Antitumor Activity in Nude Mouse Tumor Xenograft Model

Tumor cells can be harvested, suspended in PBS, and can be injected s.c.into the right and left flanks (2×10⁶ cells/flank) of 8-week-old femalenude mice as reported previously (Sun, J., Blaskovic, et al. CancerRes., 59: 4919-4926, 1999). When tumors reach about 100-150 mm³, animalsare randomized and dosed i.p. with 0.2 ml vehicle of the TCN, TCN-P,TCN-PM and/or related compounds and/or the taxane daily. Control animalsreceive DMSO (20%) vehicle, whereas treated animals can be injected withAPI-2 (1 mg/kg/day) in 20% DMSO.

API-2 Inhibits the Growth of Tumors in Nude Mice that Overexpress Akt.Frequent overexpression/activation and/or amplification of AKT1 and AKT2in human ovarian and pancreatic cancer was shown (Cheng, J. Q., andNicosia, S. V. AKT signal transduction pathway in oncogenesis. In SchwabD, Editor, Encyclopedic Reference of Cancer. Berlin Heidelberg and NewYork: Springer; 2001. pp 35-7). Inhibition of Akt pathway by inhibitorsof PI3K, HSP70, Src and farnesyltransferase resulted in cell growtharrest and induction of apoptosis (Solit, D. B., et al. Cancer Res., 63:2139-2144, 2003, Xu. W., ct al. Cancer Res., 63: 7777-7784, 2003). Arecent study showed that the tumor growth of xenografts with elevatedAkt was also significantly inhibited by intratumoral injection ofadenovirus of dominant negative Akt (Jetzt, A., et al. Cancer Res. 63:697-706, 2003). Because API-2 inhibits Akt signaling and inducesapoptosis and cell growth arrest only in cancer cells with elevatedlevels of Akt (FIG. 3), the growth of tumors with elevated levels of Aktshould be more sensitive to API-2 than that of tumors with low levels ofAkt in nude mice. To this end, s.c. Akt-overexpressing cells (OVCAR3,OVCAR8 and PANC-1) are s.c. implanted into the right flank, and thosecell lines that express low levels of Akt (OVCAR5 and COLO357) into theleft flank of mice. When the rumors reach an average size of about100-150 mm³, the animals are randomized and treated i.p. with eithervehicle or API-2 (1 mg/kg/day). As illustrated in FIG. 4B, OVCAR-5 andCOLO357 tumors treated with vehicle grew to about 800-1,000 mm³ 49 daysafter tumor implantation. OVCAR3, OVCAR8 and PANC1 tumors treated withvehicle control grew to about 700-900 mm³ 49 days after tumorimplantation. API-2 inhibited OVCAR3, OVCAR8 and PANC1 tumor growth by90%, 88% and 80%, respectively. In contrast, API-2 has little effect onthe growth of OVCAR5 and COLO357 cells in nude mice (FIGS. 4B-4D anddata not shown). At dose 1 mg/kg/day, API-2 had no effects on bloodglucose level, body weight, activity and food intake of mice. In treatedtumor samples, Akt activity was inhibited by API-2 without change oftotal Akt content (FIG. 4E). Taken together, these results indicate thatAPI-2 selectively inhibits the growth of tumors with elevated levels ofAkt.

8.3 Example 3 TCN Directly Inhibits Wild Type Akt Kinase Activity

API-2 (TCN) can directly inhibit wild type Akt kinase activity inducedby PDK1 in vitro (FIG. 1). This result supports that API-2 is a directAkt inhibitor and that the underlying mechanism may be API-2 binding toPH domain and/or threonine-308 of Akt. An in vitro kinase assay isperformed with recombinant of PDKI and Akt in a kinase buffer containingphosphatidylinositol-3,4,5-P3 (PIP3), API-2 and histone H2B assubstrate. After incubation of 30 min, the reactions were separated bySDS-PAGE and exposed in a film.

8.4 Example 4 TCN is Effective in Cancer Resistant Cells

The effects of TCN (API-2) are tested in cisplatin, paclitaxel, andtamoxifen resistant A270CP, C-13, OVCAR433 and MCF7/TAM cells. API-2overcame cisplatin, paclitaxel, and tamoxifen resistance in these cells

This invention has been described with reference to its preferredembodiments. Variations and modifications of the invention, will beobvious to those skilled in the art from the foregoing detaileddescription of the invention. It is intended that all of thesevariations and modifications be included within the scope of thisinvention.

8.5 Example 5 TCN Potentiates Growth Inhibition by Trastuzumab andInduces Apoptosis

Materials and Methods

Cell Lines and Cell Cultures. The tumorigenic BT474.m1 sublinemaintained in Dulbecco's modified Eagle's medium: Ham's F-12 medium(1:1) with 8-10% FBS.

Antibodies and Reagents. Trastuzumab was a gift from Genentech (SanFrancisco, Calif.). RAD0000 (everolimus) was a gift from Novartis (EastHanover, N.J.). QLT0267 and KP 372-1 were gifts from QLT Inc.(Vancouver, BC). Triciribine(6-Amino-4-methyl-8-((3-D-ribofuranosyl)-4H,8H-pyrrolo[4,3,2-de]pyrimido[4,5-c]pyridazine)was purchased from Berry & Associates, Inc. (Ann Arbor, Mich.).Edelfosine was purchased from Calbiochem (San Diego, Calif.). Aselective Akt inhibitor, 4ADPIB(4-amino-2(3,4-dichloro-phenyl)-N-(1H-indazol-5-yl)-butyramide) (U.S.Pat. No. 6,919,340), was synthesized. PTEN antibodies were from SantaCruz Biotechnology (Santa Cruz, Calif.). 3-actin antibodies were fromSigma (St. Louis, Mo.). All other antibodies were purchased from CellSignaling Technology (Danvers, Mass.).

PTEN Antisense and Non-specific Oligonucleotide Transient Transfection.Antisense (AS) oligonucleotides specific for PTEN, control non-specific(NS) oligonucleotides and procedures for transfection were followedaccording to Nagata Y. et al., Cancer Cell, 2004. 6(2): p. 117-27.

Cell Proliferation Assay. PTEN AS/NS transfected BT474. ml cells wereplated 2500 cells/0.32 cm² well. Cells were treated with inhibitors ofthe Akt/mTOR pathway alone or in combination with trastuzumab asdescribed for 5 days and viable cells were measured by MTS assay usingthe CellTiter 96 AQ nonradioactive cell proliferation assay kitsaccording to the manufacturer's protocol (Promega, Madison, Wis.).Treated cells were compared to control DMSO treated BT474. ml cells tocalculate percentage of growth inhibition.

APO-BRDU Tunel Assay. The PTEN AS and NS transfected BT474. ml cellswere plated in 6-well plates (4−6×10⁵ cells/well). Twenty-four hoursafter plating, the cells were treated as indicated for 72 hours withtrastuzumab, TCN and/or RAD001. The floating and adherent cells werecollected, labeled and stained using the APO-BRDU™ TUNEL assay kit(Phoenix Flow Systems, San Diego, Calif.) according to themanufacturer's protocol. Data was collected and analyzed using a FACScanflow cytometer and CellQuest Pro 4.02 software (Becton Dickinson,Franklin Lakes, N.J.). At least 10,000 events were examined.

SDS-PAGE and Immunoblot Analysis. Cells transfected with PTEN AS/NSoligonucleotides were treated as indicated. Immunoblotting was performedas described by Nagata Y. et al., Cancer Cell, 2004. 6(2): p. 117-27.

Results

Triciribine and RAD001 potentiate growth inhibition by trastuzumab inPTEN-deficient cells. To find a strategy to overcome trastuzumabresistance, particularly resistance caused by PTEN loss, 6 differentsmall molecule inhibitors were tested, which directly or indirectlytargeted the PI3K/Akt/mTOR signal transduction pathway, a major pathwayactivated by overexpression of ErbB2 and the loss of PTEN. The goal wasto identify compounds that would exhibit synergistic effects withtrastuzumab, preferably at a low dose of the compound in order tominimize toxicity. The drugs chosen targeted Akt, mTOR andintegrin-linked kinase (ILK) (FIG. 10A). BT474. ml cells are atumorigenic subline of the BT474 breast cancer cell line and expresshigh levels of ErbB2. When PTEN levels are decreased by transfectionwith PTEN antisense oligonucleotides (PTEN AS), BT474. ml breast cancercells become more resistant to the anti-proliferative effects oftrastuzumab than cells with normal levels of PTEN and provide a goodexperimental model for breast cancers in which trastuzumab resistance iscaused by PTEN loss (Nagata Y. et al., Cancer Cell, 2004. 6(2): p.117-27 and FIGS. 9B and 9C). Nonspecific oligonucleotides (NS) weretransfected as controls. Treatment with PTEN AS oligonucleotideseffectively lowered PTEN levels (FIGS. 12A and 12B), Neither PTEN AS norNS control oligonucleotides altered ErbB2 levels in the cells.

PTEN AS and NS-transfected BT474. ml cells were treated with each of the6 compounds or trastuzumab alone and in combination for 5 days andevaluated cell proliferation as compared to DMSO-treated control. Usinggrowth inhibition as a biological endpoint, we compared the ability ofeach drug to exhibit cooperative effects with trastuzumab using doses ofdrug that resulted in −20-40% growth inhibition when administered alone(FIG. 10A).

Almost all of the compounds displayed growth inhibitory effects,particularly at high concentrations and in cells with intact PTEN (FIG.10A). However, two of the compounds, triciribine and RAD001, markedlyenhanced growth inhibition in the PTEN AS cells when combined withtrastuzumab as compared to trastuzumab or either compound alone (FIG.10A). Triciribine (also called API-2), a compound that inhibits Aktactivation, potentiated growth inhibition by trastuzumab over a 20-foldconcentration range (FIG. 10B). The mTOR inhibitor RAD001 (everolimus)increased growth inhibition by trastuzumab when RAD001 was administeredat low doses (<1 nM) (FIG. 10C). Strikingly, triciribine and RAD001 wereable to cooperate with trastuzumab to inhibit cell growth at similarlevels in the PTEN AS and NS cells (FIGS. 10B and 10C). In essence,triciribine and RAD001 were able to restore trastuzumab sensitivity toPTEN-deficient cells. Triciribine and RADOO1 were also effective assingle agents, both in PTEN AS and NS cells, at doses greater than 5 mMand 1.5 nM for triciribine and RAD001, respectively (FIGS. 10B and 10C).

A third compound, the ILK inhibitor QLT0267, potentiated growthinhibition by trastuzumab within a narrow dose range (−5-15 pM)(FIG.10A). Because the dose range in which QLT0267 exhibited cooperativeeffects with trastuzumab was narrow, this compound further was notinvestigated further. At concentrations greater than 20 pM, QLT0267 hadno cooperative effect with trastuzumab but significantly inhibited cellgrowth as a single agent.

Induction of Apoptosis Following Combination Treatment. To assess ifgrowth inhibition was accompanied by apoptosis, we treated PTEN AS andNS transfected BT474. ml cells with triciribine, RAD001 and trastuzumab,alone or combined, and quantified the levels of apoptosis (FIG. 10).RAD001 did not significantly induce apoptosis alone or in combinationwith trastuzumab. Although the number of TUNEL-positive, apoptotic cellsincreased slightly following treatment with trastuzumab or triciribinealone, this increase was not statistically significant. However, thecombination of triciribine and trastuzumab, significantly inducedapoptosis as compared with all other treatments in both PTEN AS and NStransfected cells (FIG. 11).

8.6 Example 6 TCN Inhibits Activation of Akt and mTOR Inhibition ofDownstream Signaling Molecules

Immunoblot analysis verified that triciribine and RAD001 blockedactivation of Akt and mTOR, important signaling molecules activated byErbB2 and the targets of triciribine and RAD001 respectively.Phosphorylation of Akt on Thr308 and Ser473 was analyzed as an indicatorof Akt activity and mTOR activity was assessed by the phosphorylation ofp70S6K (70-kDa ribosomal protein S6 kinase), an mTOR target. Aftertriciribine treatment, phosphorylation of Akt on both sites wassubstantially decreased (FIG. 12A). In PTEN-deficient cells, the levelsof Akt phosphorylation following triciribine and trastuzumab combinationtreatment were similar to those seen in cells with intact PTEN (FIG.12A, lanes 4&8). Thus, triciribine overcame the adverse effects of PTENloss by effectively blocking Akt activation. RAD001 dramatically blockedphosphorylation of p70S6K (FIG. 12B). However, RAD001 combined withtrastuzumab did not lower p70S6K phosphorylation beyond that seen withRAD001 alone (FIG. 12B). A feedback loop has been recently identifiedwhich results in Akt phosphorylation and activation following treatmentwith mTOR inhibitors, such as RAD001 (O'Reilly K. E. et al., Cancer Res,2006. 66(3): p. 1500-8). It was also observed that feedback activationof Akt by RAD001 and combination therapy with trastuzumab and RAD001eliminated Akt phosphorylation by this feedback loop (FIG. 12B, lanes 3vs. 4), consistent with the notion that Akt activation following mTORinhibition is dependent on upstream receptor tyrosine kinases (O'ReillyK. E. et al., Cancer Res, 2006. 66(3): p. 1500-8). In summary, bothdrugs inhibited their predicted target kinases and combination treatmenthad a larger inhibitory effect on the Akt/mTOR signaling pathway thanany single agent, even in PTEN-deficient cells.

8.7 Example 7 TCN and Trastuzumab Inhibit Tumor Growth in PTEN-deficientTumors

Materials and Methods

Xenograft Human Tumor Model in SCID Mice. Female, 6-week-old, severecombined immunodeficiency (SCID) mice were from Taconic Farms (Hudson,N.Y.). Tumor xenografts were performed as described in Nagata Y. et al.,Cancer Cell, 2004. 6(2): p. 117-27. When the xenograft tumors reachedthe average size of 100-150 mm³, the mice were divided into 6 groups,each with 7 mice and an even distribution of tumor sizes, and treated asfollows. PTEN antisense (30 pg) oligonucleotides were administered toeach mouse weekly via intratumor injection. One week after PTEN ASoligonucleotide administration was initiated, drug treatment began.Trastuzumab was given at a dose of 0.5 mg/kg twice a week in 200 pLsaline through intratumor injection at multiple sites. Triciribine wasgiven at a dose of 0.5 mg/kg/day in 200 pL 20% DMSO saline solutionthrough intraperitoneal (I.P.) injection. RAD001 was given via gavage ata close of 1 pg/kg in 500 pL 5% glucose water twice a week. 20% DMSOsaline solution (200 pL/day) was given through I.P. injection. Thetumors were measured twice weekly with calipers and the volume of thetumors was calculated as: volume=length×width²/2.

Statistical Analysis. One-way ANOVA was performed using GraphPad Prism3.0 for Windows (Graph Pad Software, San Diego, Calif.).

Results

The earlier biological and molecular data were very promising, however,in vivo studies provide the most stringent test for therapeuticefficacy. Therefore, triciribine and RAD001 were tested in vivo. BT474.ml cell xenografts were injected into the mammary fat pad of 6-week-oldSCID mice. After tumors formed, the mice received PTEN AS weekly viaintratumor injection. This protocol effectively models PTEN-deficienttumors in vivo (Nagata Y. et al., Cancer Cell, 2004. 6(2): p. 117-27).The mice were randomized into treatment groups receiving triciribine,RAD001, trastuzumab or DMSO alone or in combination. After treatment,the growth patterns of the tumors treated with DMSO, trastuzumab,RAD001, or triciribine alone were similar (FIGS. 13A and 13B). Growth ofthe tumors was not inhibited and the mice were euthanized after 3 weeksdue to large rumor burdens. In contrast, combination treatment withtriciribine and trastuzumab dramatically and significantly inhibitedtumor growth (FIG. 13A). Many of the tumors actually decreased in sizeand four of 7 mice had no palpable tumors after 5 weeks of treatment.Following treatment with RAD001 and trastuzumab, tumor growth wasrelatively slower compared to RAD001 or trastuzumab alone (FIG. 13B).Thus combining trastuzumab with triciribine or RAD001 effectivelyinhibited ErbB2-overexpressing, PTEN-deficient human breast cancerxenografts in vivo.

This invention has been described with reference to its preferredembodiments. Variations and modifications of the invention will beobvious to those skilled in the art from the foregoing detaileddescription of the invention. It is intended that all of thesevariations and modifications be included within the scope of thisinvention.

Immunoblot analysis verified that triciribine and RAD001 blockedactivation of Akt and mTOR, important signaling molecules activated byErbB2 and the targets of triciribine and RAD001 respectively.Phosphorylation of Akt on Thr308 and Ser473 was analyzed as an indicatorof Akt activity and mTOR activity was assessed by the phosphorylation ofp70S6K (70-kDa ribosomal protein S6 kinase), an mTOR target. Aftertriciribine treatment, phosphorylation of Akt on both sites wassubstantially decreased (FIG. 12A). In PTEN-deficient cells, the levelsof Akt phosphorylation following triciribine and trastuzumab combinationtreatment were similar to those seen in cells with intact PTEN (FIG.12A, lanes 4&8). Thus, triciribine overcame the adverse effects of PTENloss by effectively blocking Akt activation. RAD001 dramatically blockedphosphorylation of p70S6K (FIG. 12B). However, RAD001 combined withtrastuzumab did not lower p70S6K phosphorylation beyond that seen withRAD001 alone (FIG. 12B). A feedback loop has been recently identifiedwhich results in Akt phosphorylation and activation following treatmentwith mTOR inhibitors, such as RAD001 (O'Reilly K. E. et al., Cancer Res,2006. 66(3): p. 1500-8). It was also observed that feedback activationof Akt by RAD001 and combination therapy with trastuzumab and RAD001eliminated Akt phosphorylation by this feedback loop (FIG. 12B, lanes 3vs. 4), consistent with the notion that Akt activation following mTORinhibition is dependent on upstream receptor tyrosine kinases (O'ReillyK. E. et al., Cancer Res, 2006. 66(3): p. 1500-8). In summary, bothdrugs inhibited their predicted target kinases and combination treatmenthad a larger inhibitory effect on the Akt/mTOR signaling pathway thanany single agent, even in PTEN-deficient cells.

8.8 Example 8 Antitumor Activity in the Nude Mouse Tumor Xenograft Model

Tumor cells can be harvested, suspended in PBS, and can be injected s.c.into the right and left flanks (2×10⁶ cells/flank) of 8-week-old femalenude mice as reported previously (Sun, J., Blaskovic, et al. CancerRes., 59: 4919-4926, 1999). When tumors reach about 100-150 mm³, animalsare randomized and dosed i.p. with 0.2 ml vehicle of the TCN, TCN-P,TCN-PM and/or related compounds and/or anthracycline analogs daily.Control animals receive DMSO (20%) vehicle, whereas treated animals canbe injected with API-2 (1 mg/kg/day) in 20% DMSO.

8.9 Example 9 Antitumor Activity in the Nude Mouse Tumor Xenograft Model

Tumor cells can be harvested, suspended in PBS, and can be injected s.c.into the right and left flanks (2×10⁶ cells/flank) of 8-week-old femalenude mice as reported previously (Sun et al., 1999, Cancer Res 59:4919-4926). When tumors reach about 100-150 mm³, animals are randomizedand dosed i.p. with 0.2 ml vehicle of the TCN, TCN-P, TCN-PM and/orrelated compounds and/or one or more platinum compounds daily. Controlanimals receive DMSO (20%) vehicle, whereas treated animals can beinjected with API-2 (1 mg/kg/day) in 20% DMSO.

API-2 Inhibits the Growth of Tumors in Nude Mice that Overexpress Akt.Frequent overexpression/activation and/or amplification of AKT1 and AKT2in human ovarian and pancreatic cancer was shown (Cheng et al., AKTsignal transduction pathway in oncogenesis, in Schwab D(ed.)Encyclopedic Reference of Cancer, Springer, pp 35-7). Inhibition of Aktpathway by inhibitors of PI3K, HSP70, Src and farnesyltransferaseresulted in cell growth arrest and induction of apoptosis (Solit et al.,2003, Cancer Res 63: 2139-2144; Xu et al., 2003, Cancer Res 63:7777-7784). A recent study showed that the tumor growth of xenograftswith elevated Akt was also significantly inhibited by intratumoralinjection of adenovirus of dominant negative Aki (Jetzt et al., 2003,Cancer Res 63: 697-706). Because API-2 inhibits Akt signaling andinduces apoptosis and cell growth arrest only in cancer cells withelevated levels of Akt (FIG. 3), the growth of tumors with elevatedlevels of Akt should be more sensitive to API-2 than that of tumors withlow levels of Akt in nude mice. To this end, s.c. Akt-overexpressingcells (OVCAR3, OVCAR8 and PANC-1) are s.c. implanted into the rightflank, and those cell lines that express low levels of Akt (OVCAR5 andCOLO357) into the left flank of mice. When the tumors reach an averagesize of about 100-150 mm³, the animals are randomized and treated i.p.with either vehicle or API-2 (1 mg/kg/day). As illustrated in FIG. 4B,OVCAR-5 and COLO357 tumors treated with vehicle grew to about 800-1,000mm³ 49 days after tumor implantation. OVCAR3, OVCAR8 and PANC1 tumorstreated with vehicle control grew to about 700-900 mm³ 49 days aftertumor implantation. API-2 inhibited OVCAR3, OVCAR8 and PANC1 tumorgrowth by 90%, 88% and 80%, respectively. In contrast, API-2 has littleeffect on the growth of OVCAR5 and COLO357 cells in nude mice (FIGS.4B-4D and data not shown). At dose 1 mg/kg/day, API-2 had no effects onblood glucose level, body weight, activity and food intake of mice. Intreated tumor samples, Akt activity was inhibited by API-2 withoutchange of total Akt content (FIG. 4E). Taken together, these resultsindicate that API-2 selectively inhibits the growth of tumors withelevated levels of Akt.

8.10 Example 10 TCN Overcomes Cisplatin Resistance in CisplatinResistant Ovarian Cancer Cells

Ovarian cancer cell lines sensitive to cisplatin, A2780S and OV2008, andtheir cisplatin resistant derivatives, A2780CP and C13, respectively,were treated with 0, 10, 20 and 30 μM cisplatin, followed by anassessment of the percentage of cells surviving the cisplatin. Theresistance to cisplatin toxicity was confirmed in the A2780S and C13cell lines (FIG. 14A). Lysates from all four cell types were resolved bySDS-PAGE and probed with antibodies to phosphorylated Akt and total Akt.The cisplatin resistant cell lines endogenously express significantlyhigher levels phosphorylated Akt (FIG. 14B).

It was next assessed what effects TCN would have on cisplatinresistance. A2780CP and C13 ovarian cancer cells were treated with 0, 5,10 and 20 μM, with or without 10 μM TCN, followed by measurement of cellsurvival. The addition of TCN significantly lowered the resistance tocisplatin in both cell lines (FIG. 15). C13 cells were then treated with0, 1, 5, 10 and 20 μM TCN with or without 10 μM cisplatin, followed bymeasurement of cell survival. The combination of TCN and cisplatinsynergistically reduced cell survival (FIG. 16).

C13 cells were injected subcutaneously in nude mice, followed bytreatment with vehicle (DMSO), cisplatin alone (2 mg/kg/day), TCN alone(2 mg/kg/day) or TCN and cisplatin. Tumor mass was assessed at weeklyintervals for four weeks. The combination of cisplatin and TCN enhancedthe ability to suppress the growth and progression of the tumor (FIG.17).

8.11 Example 11 TCN Enhances the Effects of mTOR Inhibition

OV3 and MCF7 cells were treated with the mTOR inhibitors rapamycin andRAD001, respectively, over a 24 hour time course. Lysyates were resolvedby SDS-PAGE and transferred to a membrane for western blotting.Membranes were probed with antibodies to phospho-Akt, total Akt,phospho-p70S6K, and total p70S6K. Membranes with lysates derived fromOV3 cells were additionally probed with antibodies to phospho-FKHRL 1and total FKHRL 1. The use of mTOR inhibitors lead to rapid activationof Akt pathway in both cell lines (FIG. 18).

OV3 cells were then treated with either control, rapamycin, or rapamycinand TCN and MCF7 cells were treated with control. RAD001, or RAD001 andTCN. TCN markedly decreased the mTOR inhibitors' increase in Aktphosphorylation without a subsequent increase in p70S6K activity (FIG.19). DU-145, MCF7 and OV3 cell growth rates were then assessed over 6days in the presence of control, mTOR inhibitor, TCN, or TCN and mTORinhibitor. The inhibition on cell growth by mTOR inhibition was enhancedby TCN (FIG. 20).

8.12 Example 12 TCN Overcomes Aurora-A Induced Cisplatin Resistance

Auruoa-A is a serine/threonine kinase involved in progression of thecell cycle. The effect of Aurora-A on sensitivity to cisplatin wasexamined. A2780S and A2780CP cells, as well as OV2008 cells, weretransfected with empty vector or with a construct encoding an HA-taggedAurora-A protein. Expression of Aurora-A was confirmed by westernblotting lysates resolved by SDS-PAGE and transferred to a membrane(FIG. 21, top panels). The viability of cells was assessed in thepresence of paclitaxel and cisplatin. A2780S and OV2008 cellstransfected with Auroa-A showed improved viability over A2780S andOV2008 cells transfected with empty vector only (FIG. 21).

It was next assessed what effect Aurora-A exhibited on the induction ofapoptosis induced by cisplatin. A2780S cells were transfected with emptyvector or with a construct encoding the Aurora-A protein. Cells werethen treated with cisplatin. Immunofluorescent staining (FIG. 22A) showsthat expression of Aurora-A inhibits cytochrome c release. Western blotanalysis revealed that introduction of Aurora-A increasesphosphorylation of Akt, as well as Akt kinase activity (FIGS. 23B andC).

Next, A2780S and OV2008 cells, transfected with either empty vector orAurora-A, were treated with either cisplatin, TCN, or cisplatin and TCN.Both A2780S and OV2008 cells expressing Aurora-A showed markedlydecreased resistance to cisplatin in the presence of TCN (FIG. 23).

This invention has been described with reference to its preferredembodiments. Variations and modifications of the invention will beobvious to those skilled in the art from the foregoing detaileddescription of the invention. It is intended that all of thesevariations and modifications be included within the scope of thisinvention.

What is claimed is:
 1. A diagnostic method for treating a subject havinga tumor or cancer, which tumor or cancer overexpresses AKT kinasecomprising: (i) obtaining a biological sample from the subject; (ii)determining whether the sample overexpresses AKT kinase; (iii) if saidsample overexpresses AKT kinase, a. administering to said subject: i. atleast one compound of formula I selected from the group consisting ofthe following compounds:

wherein each R₂′, R₃′, and R₅′ is independently hydrogen; optionallysubstituted phosphate or phosphonate; mono-, di-, or triphosphate; acyl;lower acyl; alkyl; lower alkyl; amide; sulfonate ester; alkyl sulfonateester; arylalkyl sulfonate ester; sulfonyl; methanesulfonyl; benzylsulfonyl, wherein the phenyl group of said benzyl is optionallysubstituted with one or more halo, hydroxyl, amino, alkylamino,arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate,phosphoric acid, phosphate, or phosphonate; optionally substitutedarylsulfonyl; a lipid; phospholipid; an amino acid; a carbohydrate; apeptide; or cholesterol; or other pharmaceutically acceptable leavinggroup that, in vivo, provides a compound of said formula I wherein R₂′,R₃′ or R₅ ′ is independently H or mono-, di- or tri-phosphate; whereinR^(x) and R^(y) are indepenthmtly hydrogen; optionally substitutedphosphate; acyl; lower acyl; amide; alkyl; lower alkyl; aromatic;polyoxyalkylenc; polyethyleneglycol; optionally substitutedarylsulfonyl; a lipid; a phospholipid; an amino acid; a carbohydrate; apeptide; or cholesterol; or other pharmaceutically acceptable leavinggroup; and wherein R₁ and R₂ each are independently H, optionallysubstituted straight chained, branched or cyclic alkyl, lower alkyl,alkenyl, or alkynyl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl orheteroaryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonyl, alkylsulfonyl, arylsulfonyl, or aralkylsulfonyl; ii.trastuzumah or a salt thereof; and iii. a pharmaceutically acceptablecarrier.
 2. The composition of claim 1, wherein the compound of formulaI is triciribine.
 3. The composition of claim 1, wherein the compound offormula I is triciribine phosphate.
 4. The composition of claim 1,wherein the compound of formula I is triciribine phosphonate.
 5. Thecomposition of claim 1, wherein the compound of formula I is present ina dose amount of at least 20 mg/m².
 6. The composition of claim 1,wherein the compound of formula I is present in an amount of at least 10mg/m².
 7. The composition of claim 1, suitable for parenteraladministration.
 8. The composition of claim 1, wherein the parenteraladministration is intravenous administration.
 9. The composition ofclaim 1, suitable for oral administration.
 10. The composition of claim1, suitable for topical administration.
 11. The composition of claim 1,wherein the trastuzumab or a salt thereof is present in an amount fromabout 1 mg to about 1000 mg.
 12. The composition of claim 1, wherein thetrastuzumab or a salt thereof is present in an amount from about 100 mgto about 500 mg.
 13. The composition of claim 1, wherein the trastuzumabor a salt thereof is present in an amount from about 200 mg to about 450mg.
 14. The composition of claim 1, wherein the trastuzumab or a saltthereof is present in an amount of about 440 mg.
 15. The composition ofclaim 1, wherein the administration of a compound of formula I andtrastuzumab is a single composition.
 16. The composition of claim 1,wherein the administration of a compound of formula I and trastuzumab isconcurrently administered.
 17. The composition of claim 1, wherein theadministration of a compound of formula I is followed by theadministration of trastuzumab.
 18. The composition of claim 1, whereinthe administration of trastuzumab is followed by the administration of acompound of formula I.